Reducing the Risk of Pathological Effects of Traumatic Brain Injury

ABSTRACT

The present disclosure provides methods and compositions for reducing the risk of pathological effects of traumatic brain injury.

This application claims the benefit of the filing date of U.S. Appl. No.61/251,230, filed Oct. 13, 2009, the entirety of which is fullyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Traumatic brain injury (TBI) is a head injury caused by trauma to thebrain. The damage can be confined to one area of the brain (focal) orinvolve more than one area of the brain (diffuse). TBI can be mild,moderate or severe. While some symptoms appear immediately, others donot appear until days, weeks, months or even years after the TBIevent(s). Symptoms of mild TBI include headache, confusion, dizziness,blurred vision, changes in mood, and impairment in cognitive function,such as memory, learning, and attention. Symptoms of moderate to severeTBI include, in addition to those observed for mild TBI, nausea,convulsions or seizures, slurring of speech, numbness of extremities,and loss of coordination.

2. Background Art

Following a traumatic injury to the central nervous system (CNS), acascade of physiological events can lead to neuronal loss including, forexample, an inflammatory immune response and excitotoxicity resultingfrom the initial impact disrupting the glutamate, acetylcholine,cholinergic, GABA_(A), and NMDA receptor systems. In addition, thetraumatic CNS injury is frequently followed by brain edema that enhancesthe cascade of injury and leads to further secondary cell death andincreased patient mortality.

Although major brain injury is often associated with cerebral hemorrhageor swelling, many instances have diffuse damage to neurons and theirconnecting fibers. Experiments employing anterograde tracers haverevealed that traumatic axonal injury is a progressive event involving afocal impairment of axoplasmic transport leading to axonal swelling andultimate disconnection in the hours to days following TBI (Raghupathi Ret al., J Neurotrauma. 17:927-38 (2000)). Initial disruption of the axonplasma membrane results in ion channel dysregulation and loss of calciumhomeostasis. Subsequently, a series of calcium dependent cascades areactivated, resulting in mitochondrial damage and cytochrome c release.Ultimately, cytochrome c release may activate a caspase-3 mediatedapoptotic cascade of proteolytic cleavage of cytoskeletal substratesresulting in the axonal disconnection characteristic of traumatic axonalinjury (Wang et al., Science 284:5412 339-343 (1999); Buki et al., J.Neurosci. 20:2825-2834 (2000); Eldadah et al., J. Neurotrauma. 17:10811-829 (2000)).

Traditional concepts of TBI also involve primary and secondary injuryphases. The primary injury is represented by the moment of impact,resultant from the impartation of kinetic energy and force vectors ineither a linear acceleration-deceleration or rotatory fashion, or acombination of both. In addition to the motion of the brain within thecerebrospinal fluid space, brain contact with underlying irregularsurfaces of the skull, the establishing of micro-vacuum phenomena withinthe cerebral tissue, and the tearing and mechanical injury to neuronsand particularly their projections can result in both local and remotedamage. At the clinical level, treatment attempts to minimize secondaryinjury by preventing or treating hypotension, hypoxia, and edema.

A tertiary phase of TBI includes what are now recognized as ongoingabnormalities in glucose utilization, cellular metabolism, as well asmembrane fluidity, synaptic function, and structural integrity (Hovda,Crit Care Med. 35:663-4 (2007); Aoyama et al, Brain Res. 1230:310-9(2008), published electronically Jul. 9, 2008). In general, axonmembranes are injured, ionic leakage occurs and axonal transport isinterrupted in a progressive manner. This concept is reinforced byrecent autopsy findings in professional contact sports athletes showingmulti-focal areas of damaged neurons and their processes, remarkable fortau antibody staining, believed to represent numerous times and regionsof injury from multiple concussions (Omalu et al., Neurosurgery57:128-34 (2005); Omalu et al., Neurosurgery 59:1086-92 (2006)).

Treatment of traumatic brain injury have included diuretics,anti-convulsants, and AMPA/NMDA receptor antagonists. However, it isdesirable to have treatments that can provide a prophylacticneuroprotective effect that can reduce the risk of neurological damageassociated with traumatic brain injury, particularly in light of thecomplex physiological cascade of events that follow the initial insultin traumatic brain injury.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides a method for prophylactic treatment thatreduces the risk of pathological effects associated with traumatic braininjury. In the embodiments herein, a composition comprising DHA isadministered to a subject at risk for traumatic brain injury prior tothe subject engaging in an activity associated with a risk of traumaticbrain injury.

In some embodiments, the method comprises administering to a subject whois at risk of traumatic brain injury a composition comprisingdocosahexaenoate (DHA), wherein the composition is administered in aprophylactically effective amount for a sufficient time period prior toengagement in an activity associated with a risk of traumatic braininjury to reduce the risk of pathological effects of traumatic braininjury.

In some embodiments, the method for reducing the risk of pathologicaleffects of traumatic brain injury, comprises: (a) selecting a subjectwho is at risk of traumatic brain injury; and (b) administering to thesubject a composition comprising DHA, wherein the composition isadministered in a prophylactically effective amount for a sufficienttime period prior to engagement in an activity associated with a risk oftraumatic brain injury to reduce the risk of pathological effects oftraumatic brain injury.

In the embodiments herein, the composition comprising DHA can be anysource in which DHA is present in sufficient amounts for administrationto achieve a prophylactic therapeutic effect. In some embodiments, theDHA-containing composition comprises fish oil, including, among others,fish oil enriched in omega-3 fatty acids, such as oil from tuna,sardines, anchovies, and mackerel. In some embodiments, theDHA-containing composition comprises primrose oil, flaxseed oil, canolaoil, walnut oil, and sunflower oil. In some embodiments, combination ofoils from different sources can be used to prepare the composition. TheDHA from these sources can be prepared in the form of alkylesters,triglycerides, or free fatty acids.

In some embodiments, the composition has a DHA to EPA ratio of greaterthan 4:1. In some embodiments, the DHA to EPA ratio is at least 10:1 orat least 100:1.

In embodiments where the DHA is in the form of an alkylester, thecontent of the DHA (in the form of an alkylester) is at least about 85wt % of the total fatty acid content of the composition. In someembodiments, the alkylester composition has a DHA content of about 85 toabout 96 wt % of the total fatty acid content. In some embodiments, thealkylester composition has a DHA content of about 85 to 96 wt % of thetotal fatty acid content, and an EPA content of about 0.1 wt % or lessof the total fatty acid content.

In some embodiments of the method, the composition has a DHA content ofat least about 40 wt % of the total fatty acid content. In someembodiments, the composition has a DHA content of about 40 to about 50wt % of the total fatty acid content. In certain embodiments, thecomposition has a DHA content of about 40 to about 50 wt % of the totalfatty acid content, and an EPA content of about 3 wt % or less, or 2 wt% or less, of the total fatty acid content. In certain embodiments, theEPA content is less than 0.2 wt % of the total fatty acid content.

In some embodiments, the composition has a DHA content of at least about55 wt % of the total fatty acid content. In some embodiments,composition has a DHA content of about 55 to 65 wt % of the total fattyacid content. In some embodiments, the composition has a DHA content ofabout 55 to 65 wt % of the total fatty acid content, and an EPA contentof less than 0.2 wt % of the total fatty acid content.

In some embodiments, the composition comprising the DHA is a microbialoil or derived from a microbial oil, such as from Crypthecodinium cohniior Schizochytrium sp.

In some embodiments herein, the subject to be treated is at risk for aclosed head injury, such as a concussion or contusion. A subject at riskfor such injury can include, among others, a subject participating in anathletic event with occurrence of concussions. Exemplary subjects inthis category include, among others, football players, boxers, andhockey players.

In some embodiments, the subject to be treated is at risk for apenetrating head injury. A subject at risk for a penetrating head injurycan include, among others, a combatant in an armed conflict, forexample, a soldier.

In some embodiments, a prophylactically effective amount of DHA isadministered to the subject for a sufficient time period prior to ananticipated engagement in an activity associated with a risk oftraumatic brain injury. In some embodiments, a prophylacticallyeffective amount of DHA is administered for at least about 28 days priorto the anticipated engagement in the activity that is associated with arisk of traumatic brain injury. In some embodiments, a prophylacticallyeffective amount of DHA is administered for at least about 6 weeks priorto the anticipated engagement in the activity that is associated with arisk of traumatic brain injury. In some embodiments, a prophylacticallyeffective amount of DHA is administered for at least about two monthsprior to the anticipated engagement in the activity that is associatedwith a risk of traumatic brain injury. In some embodiments, aprophylactically effective amount of DHA is administered to the subjectfor at least about 6 weeks to about 6 months, at least about 2 to about6 months, or at least about 4 months to about 6 months prior to theanticipated engagement in the activity that is associated with a risk oftraumatic brain injury. In some embodiments, a prophylacticallyeffective amount of DHA is administered to the subject for at leastabout 2 to about 4 months prior to the anticipated engagement in theactivity that is associated with a risk of traumatic brain injury.

In the methods of the present disclosure, the subject is administered aprophylactically effective amount of DHA. In some embodiments, the DHAis administered in an amount of from about 4 mg/kg body weight/day toabout 85 mg/kg body weight/day. In some embodiments, the DHA isadministered in an amount of from about 4 mg/kg body weight/day to about60 mg/kg body weight/day; from about 5 mg/kg body weight/day to about 60mg/kg body weight/day, from about 10 mg/kg body weight/day to about 60mg/kg body weight/day, from about 20 mg/kg body weight/day to about 60mg/kg body weight/day; from about 10 mg/kg body weight/day to about 40mg/kg body weight/day; or from about 20 mg/kg body weight/day to about40 mg/kg body weight/day. In some embodiments, the DHA is administeredin an amount of about 40 mg/kg body weight/day.

In some embodiments, the invention is directed to a method of protectingthe brain of a human subject, the method comprising administering to thesubject, before an activity associated with a potential traumatic braininjuring event, an oral dosage form comprising at least 900 mg of DHA,wherein the dosage form comprising at least about 35 wt %docosahexaenoate (DHA) of the total fatty acid content, wherein thedosage form has an eicosapentaenoate (EPA) content of less than about 2wt % of the total fatty acid content. In some embodiments, the phrase“protecting the brain” refers to the prevention of the pathologicaleffects of a concussion, or the reduction of the pathological effectsassociated with a concussion, in particular, minimizing the learningand/or memory deficits associated with traumatic brain injury, e.g., aconcussion. In some embodiments, the phrase “protecting the brain”refers to an increase in brain resilience in the event of traumaticbrain injury, e.g., reducing the time required after the traumatic braininjury to reduce/eliminate any learning and/or memory deficits.

In some embodiments, the activity associated with a traumatic braininjuring event is selected from the group consisting of boxing,football, soccer, or hockey, in particular events at the high school,college, or professional level. In some embodiments, the activityassociated with a traumatic brain injuring event is selected from thegroup consisting of armed conflict or brain surgery.

In some embodiments, the invention is directed to a method of protectingthe brain of a human subject, the method comprising: (1) identifying asubject at risk of experiencing a traumatic brain injuring event, and(2) administering to the subject, before an activity associated with apotential traumatic brain injuring event, an oral dosage form comprisingat least 900 mg of DHA, wherein the dosage form comprises at least about35 wt % docosahexaenoate (DHA) of the total fatty acid content, whereinthe dosage form has an eicosapentaenoate (EPA) content of less thanabout 2 wt % of the total fatty acid content.

DETAILED DESCRIPTION OF THE INVENTION

For the descriptions herein and the appended claims, the singular forms“a”, “an” and “the” include plural referents unless the context clearlyindicates otherwise. Thus, for example, reference to “a compound” refersto more than one compound.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising,” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise.

Traumatic brain injury (TBI) is among the most frequently occurring andwidely known events that can cause brain injury and an associatedimpairment of one or more neurological functions. One of thecharacteristics of traumatic injury is the rapid and sustained increasein polyunsaturated free fatty acids and diacylglycerols (Homayoun etal., Neurochemical Research 25:269-276 (2000)). In the presentdisclosure, a DHA-containing composition is administered to a subjectprior to the traumatic brain injuring event to reduce the risk ofpathological effects of traumatic brain injury.

Accordingly, in the embodiments herein, a method for reducing the riskof pathological effects of traumatic injury can comprise administeringto a subject who is at risk of traumatic brain injury a compositioncomprising DHA, wherein the composition is administered in aprophylactically effective amount for a sufficient time period prior toengagement in an activity associated with risk of traumatic brain injuryto reduce the risk of pathological effects of traumatic brain injury.

As used herein, “traumatic brain injury” or “TBI” refers to acquiredbrain injury or a head injury, when a trauma causes damage to the brain.The damage can be focal, i.e., confined to one area of the brain, ordiffuse, involving more than one area of the brain. As used herein,“traumatic brain injury” does not include brain injury induced byischemia/reperfusion.

In some embodiments, the subject can be at risk for a closed headinjury. A “closed head injury” refers to a brain injury when the headsuddenly and violently hits an object but the object does not breakthrough the skull. In some embodiments, the closed head injury is aconcussion or contusion. A concussion is a mild form of traumatic braininjury resulting in temporary impairment of neurological function whichquickly resolves by itself, and where there are generally no grossstructural changes to the brain as the result of the condition. Anexample of a subject at risk for closed head injury includes an athleteparticipating in a sport with occurrence of concussions, such as boxing,football, or hockey.

In some embodiments, the subject can be at risk for a penetrating headinjury. A penetrating injury refers to a brain injury when an objectpierces the skull and enters brain tissue. Typically, the dura mater,the outer layer of the meninges is pierced or breached by an object,such as a high velocity projectile or objects of lower velocity such asknives, or bone fragments from a skull fracture that are driven into thebrain. An example of a subject at risk for traumatic brain injury from apenetrating head injury is a combatant in an armed conflict. In certainembodiments, the subject at risk for a penetrating head injury is apatient undergoing brain surgery.

A person of skill in the art can readily identify subjects at risk fortraumatic brain injury and administer the compositions of DHAprophylactically to reduce the risk of pathological effects of traumaticbrain injury. Thus, in some embodiments, the method for reducing therisk of pathological effects of traumatic injury can comprise: (a)selecting a subject who is at risk of traumatic brain injury; and (b)administering to the subject a composition comprising DHA, wherein thecomposition is administered in a prophylactically effective amount for asufficient time period prior to engagement in an activity associatedwith a risk of traumatic brain injury to reduce the risk of pathologicaleffects of traumatic brain injury.

In some embodiments, brain health can be improved after an activityknown to increase the likelihood of a traumatic brain injuring event,e.g., boxing, football, soccer, hockey, armed conflict, or brainsurgery, by administering the compositions of the present inventionbefore the activity. The term brain health can refer to any known methodof the maintenance or improvement of brain function by any of thestandard techniques or assessments known to those of skill in the art,including those techniques and assessments provided herein.

Various pathological effects of traumatic brain injury depend on theform of the injury and its severity. In some embodiments, thepathological effects of traumatic brain injury include immediateseizures, hydrocephalus or post-traumatic ventricular enlargement,cerebral spinal fluid leaks, vascular injuries, cranial nerve injuries,and impaired cognition (thinking, memory, and reasoning), sensoryprocessing (sight, hearing, touch, taste, and smell), communication(expression and understanding), and behavior or mental health(depression, anxiety, personality changes, aggression, acting out, andsocial inappropriateness).

In some embodiments, the pathological effect of traumatic brain injuryis postconcussion syndrome (PCS). Symptoms of PCS include headache,dizziness, vertigo, memory problems, sleep problems, and troubleconcentrating. Some patients may experience post-traumatic amnesia(PTA), either anterograde or retrograde. Anterograde PTA is impairedmemory of events that happened after the traumatic brain injury, whileretrograde PTA is impaired memory of events that happened before thetraumatic brain injury.

Many patients with mild to moderate head injuries who experiencecognitive deficits also have problems with higher level, so-calledexecutive functions, such as planning, organizing, abstract reasoning,problem solving, and making judgments, which may make it difficult toresume pre-injury work-related activities. Some may experience aphasia,defined as difficulty with understanding and producing spoken andwritten language. Other pathologies affect subtle aspects ofcommunication, such as body language and emotional, non-verbal signals.In some embodiments, the pathological effect is non-fluent aphasia, alsocalled Broca's aphasia or motor aphasia.

Long term pathological effects of traumatic brain injury include, amongothers, increased incidence of Parkinson's disease and other motorproblems; Alzheimer's disease; dementia pugilistica; and post-traumaticdementia. In fact, one effect of injury to neurons an increase inpresence of β-amyloid precursor protein, a protein associated withAlzheimer's disease.

In some embodiments, as further discussed below, an effective amount ofDHA is administered to the subject at least 6 weeks prior to thesubject's anticipated engagement in an activity associated with a riskfor traumatic brain injury. In some embodiments, an effective amount ofDHA is administered to the subject at least 2 months prior to thesubject's anticipated engagement in an activity associated with a riskfor traumatic brain injury. In some embodiments, an effective amount ofDHA is administered to the subject for at least about 2 months to about6 months or more prior to the subject's anticipated engagement in theactivity that is associated with a risk of traumatic brain injury. Insome embodiments, an effective amount of DHA is administered to thesubject for at least about 2 months to about 4 months or more prior tothe subject's anticipated engagement in the activity that is associatedwith a risk of traumatic brain injury.

In some embodiments, the subject at risk of traumatic brain injury isadministered a composition comprising docosahexaenoate or DHA. In theembodiments herein, “docosahexanoate” or refers to(all-Z)-4,7,10,13,16,19-docosahexaenoic acid, as well as any salts orderivatives thereof. Thus, the term docosahexaenoate or “DHA”encompasses the free acid DHA as well as DHA alkyl esters andtriglycerides containing DHA DHA is an ω-3 polyunsaturated fatty acid.Hence, in various embodiments, the DHA used in the method may be in theform of a phospholipid, a triglyceride, free fatty acid, and an alkylester. In some embodiments, the alkyl ester may comprise DHA methylester, ethyl ester, or propyl ester, as further described below.

In the embodiments herein, the composition comprising DHA can be anysource in which DHA is present in sufficient amounts for administrationto achieve the prophylactic therapeutic effect. These include, by way ofexample and not limitation, animal, plant and microbial sources. In someembodiments, a source of oils containing DHA suitable for thecompositions and methods described herein is an animal source. Examplesof animal sources include aquatic animals (e.g., fish; marine mammals;crustaceans such as krill and other euphausids; rotifers; etc.) andlipids extracted from animal tissues (e.g., brain, liver, eyes, etc.)and animal products such as eggs or milk. Examples of plant sourcesinclude macroalgae, flaxseeds, rapeseeds, corn, evening primrose, soyand borage. In some embodiments, the composition comprises fish oil,including, among others, fish oil enriched in omega-3 fatty acids, suchas oil from tuna, sardines, anchovies, and mackerel. In someembodiments, the DHA-containing composition comprises primrose oil,flaxseed oil, canola oil, walnut oil, or sunflower oil. The DHA fromthese sources can be in the form of alkylesters, triglycerides, or freefatty acids.

In some embodiments, the DHA-containing compositions used in the methodshas a DHA to eicosapentaenoate (EPA) ratio of at least 2:1 up to 4:1wt/wt. The term “eicosapentaenoate” or “EPA” refers to eicosapentaenoicacid, known by its chemical name (all Z) 5,8,11,14,17-eicosapentaenoicacid, as well as any salts or derivatives thereof. Thus, the term “EPA”encompasses the free acid EPA as well as EPA alkyl esters andtriglycerides containing EPA. EPA is also an ω-3 polyunsaturated fattyacid. Typical content of omega-3 fatty acids found in fatty fish have aratio of DHA to EPA ratio of 4:1 or less, wt/wt. Accordingly, the DHAcontaining composition having a DHA to EPA ratio of at least about 2:1up to 4:1 wt/wt can be obtained from fish oil, such as from tuna,sardines, anchovies, and mackerel, as noted above.

In some embodiments, the DHA containing composition used in the methodshas a DHA to EPA ratio which is higher than 4:1 wt/wt. In someembodiments of the method, the composition of DHA has a DHA to EPA ratiowhich is at least 5:1 wt/wt, at least 6:1 wt/wt, 7:1 wt/wt, at least 8:1wt/wt, at least 9:1 wt/wt, at least 10:1 wt/wt, at least 12:1 wt/wt, atleast 14:1 wt/wt, at least 16:1 wt/wt, at least 18:1 wt/wt, at least20:1 wt/wt, at least 40:1 wt/wt, at least 60:1 wt/wt, at least 80:1wt/wt, at least 100:1 wt/wt, or higher. In some embodiments of themethod, the composition of DHA has a DHA to EPA ratio of about 10:1wt/wt, 12:1 wt/wt, 14:1 wt/wt, 16:1 wt/wt, 18:1 wt/wt, 20:1 wt/wt, 40:1wt/wt, 60:1 wt/wt, 80:1 wt/wt, or 100:1 wt/wt.

In some embodiments, the composition of DHA is substantially free ofEPA. As used herein, a composition of DHA that is “substantially free ofEPA” refers to a preparation of DHA in which EPA is less than about 3 wt%, or about 2 wt %, of the total fatty acid content of the composition.Thus, in some embodiments, a composition substantially free of EPA canhave less than 2 wt % of the total fatty acid content of thecomposition, less than 1 wt % of the total fatty acid content of thecomposition, less than 0.5 wt % of the total fatty acid content of thecomposition, less than 0.2 wt % of the total fatty acid content of thecomposition, or less than 0.01 wt % of the total fatty acid content ofthe composition. In some embodiments, the EPA is not detectable in thecomposition using techniques known in the art. An exemplary techniquefor detecting the amount of EPA is direct transmethylation of the oil toform fatty acid methyl esters (FAME) followed by separation of theproducts by HPLC, gas-liquid chromatography, or gas chromatography-massspectroscopy (see, e.g., Fournier et al., J Chromatogr A. 1129:21-8(2006)). In some embodiments, the DHA composition has no EPA.

DHA can also be administered substantially free of arachidonic acid(ARA). ARA refers to the compound (all-Z)-5,8,11,14-eicosatetraenoicacid (also referred to as (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoicacid), as well as any salts or derivatives thereof. Thus, the term “ARA”encompasses the free acid ARA as well as ARA alkyl esters andtriglycerides containing ARA. ARA is an ω-6 polyunsaturated fatty acid.DHA is “substantially free of ARA” when ARA is less than about 3%(wt/wt) of the total fatty acid content of the dosage form. In someembodiments, ARA comprises less than about 2% (wt/wt) of the total fattyacid content of the dosage form, less than 1% (wt/wt) of the total fattyacid content of the dosage form, less than 0.5% (wt/wt) of the totalfatty acid content of the dosage form, less than 0.2% (wt/wt) of thetotal fatty acid content of the dosage form, or less than 0.01% (wt/wt)of the total fatty acid content of the dosage form. In some embodiments,the dosage form has no detectable amount of APA.

DHA can also be administered substantially free of docosapentaenoic acid22:5 n-6 (DPAn-6). The term “DPAn-6” refers to docosapentaenoic acid,omega 6, known by its chemical name(all-Z)-4,7,10,13,16-docosapentaenoic acid, as well as any salts oresters thereof. The term “DPAn-6” encompasses the free acid DPAn-6 aswell as DPAn-6 alkyl esters and triglycerides containing DPAn-6. DPAn-6is an ω-6 polyunsaturated fatty acid. DHA is “substantially free ofDPAn-6” when DPAn-6 is less than about 3% (wt/wt) of the total fattyacid content of the dosage form. In some embodiments, DPAn-6 comprisesless than about 2% (wt/wt) of the total fatty acid content of the dosageform, less than 1% (wt/wt) of the total fatty acid content of the dosageform, less than 0.5% (wt/wt) of the total fatty acid content of thedosage form, less than 0.2% (wt/wt) of the total fatty acid content ofthe dosage form, or less than 0.01% (wt/wt) of the total fatty acidcontent of the dosage form. In some embodiments, the dosage form has nodetectable amount of DPAn-6.

In some embodiments, the dosage form of the present invention does notcontain a measurable amount of docosapentaenoic acid 22:5n-3 (DPAn-3);docosapentaenoic acid 22:5n-6 (DPAn-6); and/or 4,7,10,13,16,19,22,25octacosaoctaenoic acid (C28:8).

In some embodiments, the DHA is administered in the substantial absenceof therapeutic levels of albumin and its pharmaceutically acceptablesalts. In some embodiments, the DHA is administered with less than 100mg, more particularly less than 10 mg, more particularly less than 5 mgand more particularly less that 1 mg of albumin and its pharmaceuticallyacceptable salts. In some embodiments, the DHA is administered with nodetectable amount of albumin.

In some embodiments, the composition of DHA may include an additionallipid. As used herein, the term “lipid” includes phospholipids (PL);free fatty acids; esters of fatty acids; triacylglycerols (TAG);diacylglycerides; monoacylglycerides; phosphatides; waxes (esters ofalcohols and fatty acids); sterols and sterol esters; carotenoids;xanthophylls (e.g., oxycarotenoids); hydrocarbons; and other lipidsknown to one of ordinary skill in the art. The lipid can be chosen tohave minimal adverse health effects or minimally affect theeffectiveness of DHA when administered in combination with DHA.

In some embodiments, the composition of DHA may include an additionalunsaturated lipid. In some embodiments, the unsaturated lipid is apolyunsaturated lipid, such as an omega-3 fatty acid or omega-6 fattyacid. An exemplary omega-6 fatty acid that may be used in thecomposition is docosapentaenoic acid (DPA), including DPAn-6 or DPAn-3.

In the methods and compositions herein, additional fatty acids can bepresent in the dosage form or unit dose or composition. These fattyacids can include fatty acids that were not removed during thepurification process, i.e., fatty acids that were co-isolated with DHAfrom an organism. In some embodiments, one or more non-DHA fatty acidscan be added to the dosage form or unit dose to achieve a desiredconcentration of specific non-DHA fatty acids. Any of these fatty acidscan be present in various concentrations. For example, in someembodiments, the dosage form or unit dose comprises 0.01% to about 4%(wt/wt) of oleic acid. In some embodiments, the dosage form or unit dosecomprises 0.01% to 0.5% (wt/wt) of one or more of the following fattyacids: (a) capric acid; (b) lauric acid; (c) myristic acid; (d) palmiticacid; (e) palmitoleic acid; (f) heptadecanoic acid; (g) stearic acid;(h) oleic acid; (i) linoleic acid; (j) α-linolenic acid; (k) arachidicacid; (l) eicosenoic acid; (m) arachidonic acid; (n) erucic acid; (o)docosapentaenoic acid 22:5n-3 (DPAn-3); and (p) nervonic acid. In someembodiments, a dosage form or unit dose comprises 0.01% to 0.1% (wt/wt)of one or more of the following fatty acids: (a) lauric acid; (b)heptadecanoic acid; (c) stearic acid; (d) arachidic acid; (e) eicosenoicacid; and (f) arachidonic acid. In some embodiments, a dosage form orunit dose comprises less than 0.5% (wt/wt) each of the following fattyacids: (a) capric acid; (b) lauric acid; (c) myristic acid; (d) palmiticacid; (e) palmitoleic acid; (f) heptadecanoic acid; (g) stearic acid;(h) linoleic acid; (i) α-linolenic acid; (j) arachidic acid; (k)eicosenoic acid; (l) arachidonic acid; (m) erucic acid; (n)docosapentaenoic acid 22:5n-3 (DPAn-3); and (o) nervonic acid. In someembodiments, the dosage form or unit doses of the present invention donot contain a measurable amount of one or more of the following fattyacids: (a) capric acid; (b) linoleic acid; (c) α-linolenic acid; and (d)docosapentaenoic acid 22:5n-3 (DPAn-3).

In some embodiments, the dosage form or unit dose comprises 0.1% to 60%(wt/wt) of one or more of the following fatty acids, or esters thereof:(a) capric acid; (b) lauric acid; (c) myristic acid; (d) palmitic acid,(e) palmitoleic acid; (f) stearic acid; (g) oleic acid; (h) linoleicacid; (i) a-linolenic acid; (j) docosapentaenoic acid 22:5n-3 (DPAn-3);(k) docosapentaenoic acid 22:5n-6 (DPAn-6); and (k)4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8). In someembodiments, the dosage form or unit dose comprises 20% to 40% (wt/wt)of one or more of the following fatty acids, or esters thereof: (a)capric acid; (b) lauric acid; (c) myristic acid; (d) palmitic acid; (e)palmitoleic acid; (f) stearic acid; (g) oleic acid; (h) linoleic acid;(i) α-linolenic acid; j) docosapentaenoic acid 22:5n-3 (DPAn-3); (k)docosapentaenoic acid 22:5n-6 (DPAn-6); and (l) 4,7,10,13,16,19,22,25octacosaoctaenoic acid (C28:8). In some embodiments, the dosage form orunit dose comprises less than 1% (wt/wt) each of the following fattyacids, or esters thereof: (a) capric acid; (b) lauric acid; (c) myristicacid; (d) palmitic acid, (e) palmitoleic acid; (f) stearic acid; (g)oleic acid; (h) linoleic acid; (1) α-linolenic acid; (j)docosapentaenoic acid 22:5n-3 (DPAn-3); (k) docosapentaenoic acid22:5n-6 (DPAn-6); and (l) 4,7,10,13,16,19,22,25 octacosaoctaenoic acid(C28:8).

In some of embodiments of DHA dosage form described herein, the dosageform is characterized by one or more the following fatty acids (oresters thereof). The embodiments provided herein may further compriseabout 2% or less (wt/wt) of capric acid (C10:0). The embodiments hereinmay further comprise about 6% or less (wt/wt) of lauric acid (C12:0).The embodiments herein may further comprise about 20% or less (wt/wt),or about 5% to about 20% (wt/wt) of myristic acid (C14:0). Theembodiments herein may further comprise about 20% (wt/wt) or less, orabout 5% to about 20% (wt/wt) of palmitic acid (C16:0). The embodimentsherein may further comprise about 3% (wt/wt) or less of palmitoleic acid(C16:1n-7). The embodiments herein may further comprise about 2% (wt/wt)or less of stearic acid (C18:0). The embodiments herein may furthercomprise about 40% (wt/wt) or less, or about 10% to about 40% (wt/wt) ofoleic acid (C18:1n-9). The embodiments herein may further comprise about5% (wt/wt) or less of linoleic acid (C18:2). The embodiments herein mayfurther comprise about 2% (wt/wt) or less of nervonic acid (C24:1). Theembodiments herein may further comprise about 3% (wt/wt) or less ofother fatty acids or esters thereof. The DHA dosage form with thepreceding characteristics may comprise DHASCO®, an oil derived fromCrypthecodinium cohnii containing docosahexaenoic acid (DHA).

An exemplary DHA (triglyceride) containing oil derived fromCrypthecodinium cohnii is characterized by the specified amount ofcomponents listed in Table 1, where “Max” refers to the amount of thecomponent that can be present up to the specified amount.

TABLE 1 Fatty Acids Concentration (wt/wt) 10:0 Max 2% 12:0 Max 6% 14:0 5% to 20% 16:0  5% to 20% 16:1 Max 3% 18:0 Max 2% 18:1 10% to 40% 18:2Max 5% 22:6 DHA 40% to 45% 24:1 Max 2% Others Max 3% ElementalComposition Arsenic Max 0.5 ppm Copper Max 0.1 ppm Iron Max 0.5 ppm LeadMax 0.2 ppm Mercury Max 0.04 ppm Phosphorous Max 10 ppm ChemicalCharacteristics Peroxide Value Max 5 Meq/Kg Free Fatty Acid Max 0.4%Unsaponifiable Matter Max 3.5%

An exemplary undiluted DHA (triglyceride) containing oil derived from

Crypthecodinium cohnii is characterized by amount of DHA describedherein, and one or more, or all of the features listed below in Table 2,where “Max” refers to the amount of the component that can be present upto the specified amount.

TABLE 2 Characteristics of Undiluted DHA Oil TEST SPECIFICATION DHACONTENT MG/DHA/G OIL MIN 480 MG/G FREE FATTY ACID MAX. 0.4% PEROXIDEVALUE (PV) MAX. 5 MEQ/KG ANISIDINE VALUE (AV) MAX 20 MOISTURE ANDVOLATILES (M & V) MAX. 0.02% UNSAPONIFIABLE MATTER MAX. 3.5% INSOLUBLEIMPURITIES MAX. 0.1% TRANS FATTY ACID MAX. 1% ARSENIC MAX. 0.5 PPMCADMIUM MAX. 0.2 PPM CHROMIUM MAX. 0.2 PPM COPPER MAX. 0.1 PPM IRON MAX.0.5 PPM LEAD MAX. 0.2 PPM MANGANESE MAX. 0.04 PPM MERCURY MAX. 0.04 PPMMOLYBDENUM MAX. 0.2 PPM NICKEL MAX. 0.2 PPM PHOSPHORUS MAX. 10 PPMSILICON MAX. 500 PPM SULFUR MAX. 100 PPM 18:1 N-9 OLEIC ACID MAX. 10%20:5 N-3 EPA MAX. 0.1% UNKNOWN FATTY ACIDS MAX. 3.0%

In some embodiments, an oil is characterized by one or more thefollowing fatty acids (or esters thereof), expressed as wt % of thetotal fatty acid content. The embodiments provided herein may furthercomprise about 2% or less (wt/wt) of capric acid (C10:0). Theembodiments provided herein may further comprise about 6% or less(wt/wt) of lauric acid (C 12:0). The embodiments provided herein mayfurther comprise about 20% or less, or about 10 to about 20% (wt/wt) ofmyristic acid (C14:0). The embodiments provided herein may furthercomprise about 15% or less, or about 5 to about 15% (wt/wt) of palmiticacid (C16:0). The embodiments provided herein may further comprise about5% or less (wt/wt) of palmitoleic acid (C16:1n-7). The embodimentsprovided herein may further comprise about 2% or less (wt/wt) of stearicacid (C18:0). The embodiments provided herein may further comprise about20% or less, or about 5% to about 20% (wt/wt) of oleic acid (C18:1n-9).The embodiments provided herein may further comprise about 2% or less(wt/wt) of linoleic acid (C18:2). The embodiments provided herein mayfurther comprise about 2% or less (wt/wt) of nervonic acid (C24:1). Theembodiments provided herein may further comprise about 3% or less(wt/wt) of other fatty acids. An oil with the preceding characteristicsmay be an oil derived from Crypthecodinium cohnii containingdocosahexaenoic acid (DHA).

In some embodiments, the dosage for, comprises, measured in percentageof free fatty acid, about 35-65%, 40-55%, 35-57%, or 57-65% DHA (22:6n-3); about 0-2% capric acid (10:0); about 0-6% lauric acid (12:0);about 10-20% myristic acid (14:0); about 5-15% palmitic acid (16:0);about 0-5% palmitoleic acid (16:1); about 0-2% stearic acid (18:0);about 5-20% or 5-25% oleic acid (18:1); about 0-2% linoleic acid (18:2);and about 0-2% nervonic acid (24:1, n-9). In one embodiment, such an oilis from a microorganism of the genus Thraustochytrium. In anotherembodiment, the free fatty acid content is less than 0.4%.

The present invention also provides compositions comprising at leastabout 40 wt % DHA and at least about 0.1 wt % of DPAn-3. In someembodiments, the compositions comprise at least about 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65 wt % DHA, optionally in triglyceride form, asa percentage of total fatty acids.

An exemplary DHA containing oil derived from Crypthecodinium cohnii ischaracterized by the specified amount of components listed in Table 3,where “Max” refers to the amount of the component that can be present upto the specified amount.

TABLE 3 FATTY ACIDS CONCENTRATION (WT/WT) 10:0 0-2% 12:0 0-6% 14:010%-20% 16:0  5%-15% 16:1 0-5% 18:0 0-2% 18:1  5%-20% 18:2 0-2%% 22:6(N-3) DHA 57%-65% 24:1 0-2% OTHERS 0-3% ELEMENTAL COMPOSITION ARSENICMAX 0.5 PPM COPPER MAX 0.1 PPM IRON MAX 0.5 PPM LEAD MAX 0.2 PPM MERCURYMAX 0.2 PPM PHOSPHOROUS MAX 10 PPM CHEMICAL CHARACTERISTICS PEROXIDEVALUE MAX 5 MEQ/KG FREE FATTY ACID MAX 0.4% UNSAPONIFIABLE MAX 3.5%MATTER TRANS FATTY ACIDS <3.5% MOISTURE AND <0.1% VOLATILES INSOLUBLE<0.1% IMPURITIES

In some embodiments, an oil is characterized by one or more thefollowing fatty acids (or esters thereof), expressed as wt % (i.e.,wt/wt) of the total fatty acid content. The embodiments provided hereinmay further comprise about 0.1% or less (wt/wt) of myristic acid (C14:0)or is not detectable. The embodiments provided herein may furthercomprise about 0.5% or less (wt/wt) of palmitic acid (C16:0). Theembodiments provided herein may further comprise about 0.5% or less(wt/wt) of palmitoleic acid (C16:1n-7). The embodiments provided hereinmay further comprise about 0.5% or less (wt/wt) of stearic acid (C18:0),or is not detectable. The embodiments provided herein may furthercomprise about 4% or less (wt/wt) of oleic acid (C18:1n-9). Theembodiments provided herein may further comprise less than 0.1% (wt/wt)of linoleic acid (C18:2) or is not detectable. The embodiments providedherein may further comprise less than 0.1% (wt/wt) of eicosapentaenoicacid (C20:5) or is not detectable. The embodiments provided herein mayfurther comprise about 2% or less (wt/wt) of decosapentaenoic acid(22:5n-3). The embodiments provided herein may further comprise about 1%or less (wt/wt) of octacosaoctaenoic acid (28:8 n-3). The embodimentsprovided herein may further comprise about 0.5% or less (wt/wt) oftetracosaenoic acid (24:1n9). The embodiments provided herein mayfurther comprise about 1% or less (wt/wt) of other fatty acids. The DHAin oil with the preceding characteristics may be in the form of a DHAester, preferably an alkyl ester, such as a methyl ester, ethyl ester,propyl ester, or combinations thereof, prepared from an algal oilprepared from the Crypthecodinium, cohnii sp.

In some embodiments of the method, the composition used has an amount ofDHA that is at least about 40 wt % of the total fatty acid content. Insome embodiments, the weight % of the DHA in the composition is at least50 wt % of the total fatty acid content, at least 55 wt % of the totalfatty acid content, at least 60 wt % of the total fatty acid content; atleast 70 wt % of the total fatty acid content; at least 80 wt % of thetotal fatty acid content; at least 85 wt % of the total fatty acidcontent; at least 90 wt % of the total fatty acid content; at least 95wt % of the total fatty acid content; at least 96 wt % of the totalfatty acid content; at least 97 wt % of the total fatty acid content; atleast 98 wt % of the total fatty acid content; or at least 99 wt % ofthe total fatty acid content. As noted above, the DHA can be in the formof alkylesters, triglycerides, or free fatty acids.

In some embodiments, DHA is present in an amount of about 35% to about99.9% (wt/wt) of the total fatty acid content of the dosage form or unitdose, about 40% to about 99% (wt/wt) of the total fatty acid content ofthe dosage form or unit dose, about 45% to about 98% (wt/wt) of thetotal fatty acid content of the dosage form or unit dose, about 65% toabout 99.9% (wt/wt) of the total fatty acid content of the dosage formor unit dose, or about 85% to about 95% (wt/wt) of the total fatty acidcontent of the dosage form or unit dose. In some embodiments, the DHA ispresent in an amount greater than about 65% (wt/wt) of the total fattyacid content of the dosage form or unit dose, greater than about 85%(wt/wt) of the total fatty acid content of the dosage foim or unit dose,greater than about 90% (wt/wt) of the total fatty acid content of thedosage form or unit dose, or greater than about 95% (wt/wt) of the totalfatty acid content of the dosage form or unit dose. In some embodiments,the oil can be diluted with other oils, such as sunflower oil, toachieve the desired concentration of fatty acids.

In some embodiments, the DHA is about 30% (wt/wt) or more of the totalfatty acid content of the dosage form or unit dose, about 30% to about99.9% (wt/wt) of the total fatty acid content of the dosage form or unitdose, about 35% to about 99.9% (wt/wt) of the total fatty acid contentof the dosage form or unit dose, about 35% to about 60% (wt/wt) of thetotal fatty acid content of the dosage form or unit dose, about 35% toabout 50% (wt/wt) of the total fatty acid content of the dosage foim orunit dose, about 37% to about 45% (wt/wt) of the total fatty acidcontent of the dosage form or unit dose, or about 38% to about 43%(wt/wt) of the total fatty acid content of the dosage form or unit dose.In some embodiments, the DHA is greater than about 35%, about 37%, about38%, about 39% or about 40% (wt/wt) of the total fatty acid content ofthe dosage form or unit dose. In some embodiments, the DHA is about 30%to about 99.5% (wt/wt) of the total fatty acid content of the dosageform or unit dose, or about 40% to about 65% (wt/wt) of the total fattyacid content of the dosage form or unit dose.

In some of these embodiments, the DHA comprises about 40% to about 45%(wt/wt) of the total fatty acid content of the dosage form or unit dose.In some of these embodiments, the DHA comprises about 35% to about 45%(wt/wt) of the total fatty acid content of the dosage form or unit dose.In some of embodiments, the DHA comprises about 55% to about 67% (wt/wt)of the total fatty acid content of the dosage form or unit dose. In someembodiments, the DHA comprises greater than about 70% (wt/wt) of thetotal fatty acid content of the dosage form or unit dose. In someembodiments, the DHA comprises about 85% to about 99.5% (wt/wt) of thetotal fatty acid content of the dosage form or unit dose.

In some embodiments, the DHA is greater than about 80% (wt/wt) of thetotal oil content of the dosage form or unit dose, about 80% to 99.9%(wt/wt) of the total oil content of the dosage form or unit dose, about85% to about 99% (wt/wt) of the total oil content of the dosage form orunit dose, about 87% to about 98% (wt/wt) of the total oil content ofthe dosage form or unit dose, or about 90% to about 97% (wt/wt) of thetotal oil content of the dosage form or unit dose. In some embodiments,the DHA is greater than about 95%, about 97%, about 98%, about 99% orabout 99.5% (wt/wt) of the total oil content of the dosage form or unitdose. With respect to comparison of DHA to total fatty acid content ortotal oil content, weight % can be determined by calculating the areaunder the curve (AUC) using standard means, e.g., dividing the DHA AUCby the total fatty acid AUC.

As used herein, “or less” or “less than about” refers to percentagesthat include 0%, or amounts not detectable by current means. As usedherein, “max” refers to percentages that include 0%, or amounts notdetectable by current means.

In some embodiments, the DHA is greater than about 80% (wt/wt) of thetotal fatty acid content of the dosage form or unit dose, about 80% to99.9% (wt/wt) of the total fatty acid content of the dosage form or unitdose, about 85% to about 99% (wt/wt) of the total fatty acid content ofthe dosage form or unit dose, about 87% to about 98% (wt/wt) of thetotal fatty acid content of the dosage form or unit dose, or about 90%to about 97% (wt/wt) of the total fatty acid content of the dosage fottnor unit dose. In some embodiments, the DHA is great than about 95%,about 97%, about 98%, about 99% or about 99.5% (wt/wt) of the totalfatty acid content of the dosage form or unit dose.

In some embodiments, the DHA comprises about 35% to about 96% of theweight of the dosage form or unit dose. In some embodiments, the DHAcomprises about 38% to about 42% of the weight of the dosage form orunit dose. In some embodiments, the DHA in the dosage form or unit dosecomprises about 35% to about 45% of the total weight of the dosage formor unit dose. In some embodiments, the DHA in the dosage form or unitdose comprises about 55% of the total weight of the dosage form or unitdose. In some embodiments, the DHA in the dosage form or unit dosecomprises about 85% to about 96% of the total weight of the dosage formor unit dose.

In some embodiments, the DHA is about 30% (wt/wt) or more of the totaloil content of the dosage form or unit dose, about 30% to about 99.9%(wt/wt) of the total oil content of the dosage form or unit dose, about35% to about 99.9% (wt/wt) of the total oil content of the dosage foamor unit dose, about 35% to about 60% (wt/wt) of the total oil content ofthe dosage form or unit dose, about 35% to about 50% (wt/wt) of thetotal oil content of the dosage form or unit dose, about 37% to about45% (wt/wt) of the total oil content of the dosage form or unit dose, orabout 38% to about 43% (wt/wt) of the total oil content of the dosageform or unit dose. In some embodiments, the DHA is greater than about35%, about 37%, about 38%, about 39% or about 40% (wt/wt) of the totaloil content of the dosage form or unit dose. In some embodiments, theDHA is about 30% to about 99.5% (wt/wt) of the total oil content of thedosage form or unit dose, or about 40% to about 65% (wt/wt) of the totaloil content of the dosage form or unit dose.

In some embodiments, the composition has a DHA content of about 40 toabout 50 wt % of the total fatty acid content. In some embodiments, thecomposition has a DHA content of about 40 to about 50 wt % of the totalfatty acid content, and an EPA content of about 3 wt % or less, or 2 wt% or less, of the total fatty acid content. In some embodiments, thecomposition has a DHA content of about 40 to 50 wt % of the total fattyacid content, and an EPA content of less than 0.2 wt % of the totalfatty acid content. In some embodiments, the composition has a DHAcontent of about 55 to 60 wt % of the total fatty acid content. In someembodiments, the composition has a DHA content of about 55 to 60 wt % ofthe total fatty acid content and an EPA content of less than about 0.2wt % of the total fatty acid content. In some of these embodiments, theDHA is in the form of a triglyceride.

In the embodiments described herein, the composition of DHA having aratio of DHA to EPA greater than 4:1 wt/wt can be obtained by standardtechniques known in the art. In some embodiments, EPA may be removedduring the purification of DHA, or alternatively, the DHA may be from anorganism that produces DHA with the levels of EPA described herein, forexample a production organism is selected that produces DHA with aninsubstantial amount of EPA. DHA can be purified to various levels. DHApurification can be achieved by any means known to those of skill in theart, and can include the extraction of total oil from an organism whichproduces DHA. In some embodiments, EPA, ARA. and/or DP An-6 are thenremoved from the total oil, for example, via chromatographic methods.Alternatively, DHA purification can be achieved by extraction of totaloil from an organism which produces DHA, but produces little, if any,amount of EPA, ARA, DPAn-6, and/or flavonoids. In some embodiments, theoil can be diluted with other oils, such as sunflower oil to achieve thedesired concentration of fatty acids.

Microbial oils useful in the present invention can be recovered frommicrobial sources by any suitable means known to those in the art. Forexample, the oils can be recovered by extraction with solvents such aschloroform, hexane, methylene chloride, methanol and the like, or bysupercritical fluid extraction. Alternatively, the oils can be extractedusing extraction techniques, such as are described in U.S. Pat. No.6,750,048 and International Pub. No. WO 2001/053512, both filed Jan. 19,2001, and entitled “Solventless extraction process,” both of which areincorporated herein by reference in their entirety. Processes for thepreparation of various forms of DHA are also described in, among others,U.S. Pub. No. 2009/0023808 “Production and Purification of Esters ofPolyunsaturated Fatty Acids” by Raman et al., and U.S. Pub. No.2007/0032548 “Polyunsaturated fatty acids for treatment of dementia andpre-dementia-related conditions” by Ellis, incorporated herein byreference.

Additional extraction and/or purification techniques are taught inInternational

Pub. No. WO 2001/076715; International Pub. No. WO 2001/076385; U.S.Pub. No. 2007/0004678; U.S. Pub. No. 2005/0129739; U.S. Pat. No.6,399,803; and International Pub. No. WO 2001/051598; all of which areincorporated herein by reference in their entirety. The extracted oilscan be evaporated under reduced pressure to produce a sample ofconcentrated oil material. Processes for the enzyme treatment of biomassfor the recovery of lipids are disclosed in International Pub. No. WO2003/092628; U.S. Pub. No. 2005/0170479; EP Pat. Pub. 0776356 and U.S.Pat. No. 5,928,696, the last two entitled “Process for extracting nativeproducts which are not water-soluble from native substance mixtures bycentrifugal force,” all of which are incorporated herein by reference intheir entirety.

In some embodiments, the DHA can be prepared as esters using a methodcomprising: a) reacting a composition comprising polyunsaturated fattyacids in the presence of an alcohol and a base to produce an ester of apolyunsaturated fatty acid from the triglycerides; and b) distilling thecomposition to recover a fraction comprising the ester of thepolyunsaturated fatty acid, optionally wherein the method furthercomprises: c) combining the fraction comprising the ester of thepolyunsaturated fatty acid with urea in a medium; d) cooling orconcentrating the medium to form a urea-containing precipitate and aliquid fraction; and e) separating the precipitate from the liquidfraction. See, e.g., U.S. Pub. No. 2009/0023808, incorporated byreference herein in its entirety. In some embodiments, the purificationprocess includes starting with refined, bleached, and deodorized oil(RBD oil), then performing low temperature fractionation using acetoneto provide a concentrate. The concentrate can be obtained bybase-catalyzed transesterification, distillation, and silica refining toproduce the final DHA product.

Methods of determining purity levels of fatty acids are known in theart, and may include, e.g., chromatographic methods such as, e.g., HPLCsilver ion chromatographic columns. Alternatively, purity levels may bedetermined by gas chromatography, with or without converting DHA to thecorresponding alkyl ester. The percentage of fatty acids may also bedetermined using Fatty Acid Methyl Ester (FAME) analysis.

In some embodiments, the DHA esters can be derived from undiluted oilfrom a single cell microorganism, and in some embodiments, fromundiluted DHASCO-T® (Martek Biosciences Corporation, Columbia, Md.). Insome embodiments, the oil from which DHA compositions can be derivedincludes single cell microorganism oils that are manufactured by acontrolled fermentation process followed by oil extraction andpurification using methods common to the vegetable oil industry. Incertain embodiments, the oil extraction and purification steps caninclude refining, bleaching, and deodorizing. In some embodiments, theundiluted DHA oil comprises about 40% to about 50% DHA by weight (about400-500 mg DHA/g oil). In certain embodiments, the undiluted DHA oil canbe enriched by cold fractionation (resulting in oil containing about 60%wt/wt of DHA triglyceride), which DHA fraction optionally can betransesterified, and subjected to further downstream processing toproduce the active DHA of the invention. In some embodiments of theinvention, downstream processing of the oil comprises distillationand/or silica refinement.

Thus, to produce oil from which DHA can be derived, in certain aspects,the following steps can be used: fermentation of a DHA producingmicroorganism; harvesting the biomass; spray drying the biomass;extracting oil from the biomass; refining the oil; bleaching the oil;chill filtering the oil; deodorizing the oil; and adding an antioxidantto the oil. In some embodiments, the microorganism culture can beprogressively transferred from smaller scale fermenters to a productionsize fermenter. In some embodiments, following a controlled growth overa pre-established period, the culture can be harvested by centrifugationthen pasteurized and spray dried. In certain embodiments, the driedbiomass can be flushed with nitrogen and packaged before being storedfrozen at −20.C. In certain embodiments, the DHA oil can be extractedfrom the dried biomass by mixing the biomass with n-hexane or isohexanein a batch process which disrupts the cells and allows the oil andcellular debris to be separated. In certain embodiments, the solvent canthen be removed.

In some embodiments, the crude DHA oil can then undergo a refiningprocess to remove free fatty acids and phospholipids. The refined DHAoil can be transferred to a vacuum bleaching vessel to assist inremoving any remaining polar compounds and pro-oxidant metals, and tobreak down lipid oxidation products. The refined and bleached DHA oilcan undergo a final clarification step by chilling and filtering the oilto facilitate the removal of any remaining insoluble fats, waxes, andsolids.

Optionally, the DHA can be deodorized under vacuum in a packed column,counter current steam stripping deodorizer. Antioxidants such asascorbyl palmitate, alpha-tocopherol, and tocotrienols can optionally beadded to the deodorized oil to help stabilize the oil. In someembodiments, the final, undiluted DHA oil is maintained frozen at −20°C. until further processing.

In some embodiments, the DHA oil can be converted to DHA ester bymethods known in the art. In some embodiments, DHA esters of theinvention can be produced from DHA oil by the following steps: coldfractionation and filtration of the DHA oil (to yield for example about60% triglyceride oil); direct transesterification (to yield about 60%DHA ethyl ester); molecular distillation (to yield about 88% DHA ethylester); silica refinement (to yield about 90% DHA ethyl ester); andaddition of an antioxidant.

In some embodiments, the cold fractionation step can be carried out asfollows:

undiluted DHA oil (triglyceride) at about 500 mg/g DHA is mixed withacetone and cooled at a controlled rate in a tank with −80° C. chillingcapabilities. Saturated triglycerides crystallize out of solution, whilepolyunsaturated triglycerides at about 600 mg/g DHA remain in the liquidstate. The solids containing about 300 mg/g can be filtered out with a20 micron stainless steel screen from the liquid stream containing about600 mg/g DHA. The solids stream can then be heated (melted) andcollected. The 600 mg/g DHA liquid stream can be desolventized with heatand vacuum and then transferred to the transesterification reactor.

In some embodiments, the transesterification step is carried out on the600 mg/g DHA oil, wherein the transesterification is done via directtransesterification using ethanol and sodium ethoxide. Thetransesterified material (DHA-ethyl ester) can then be subject tomolecular distillation and thus, further distilled (3 passes, heavies,lights, heavies) to remove most of the other saturated fatty acids andsome sterols and non-saponifiable material. The DHA-ethyl ester (DHA-EE)can be further refined by passing it through a silica column.

DHA free fatty acids (DHA-FFA) can be made using, for example, the DHAcontaining oils described above. In some embodiments, the DHA-FFA can beobtained from DHA esters. DHA triglycerides, for example, can besaponified followed by a urea adduction step to make free fatty acids.

Any source of DHA can be used in the compositions and methods describedherein, including, for example, animal, plant and microbial sources.Examples of animal sources include aquatic animals (e.g., fish, marinemammals, crustaceans, rotifers, etc.) and lipids extracted from animaltissues (e.g., brain, liver, eyes, etc.). Examples of plant sourcesinclude macroalgae, flaxseeds, rapeseeds, corn, evening primrose, soyand borage. Examples of microorganisms include microalgae, protists,bacteria and fungi (including yeast). For example, the DHA may bepurified from fish oil, plant oil, seed oil, or other naturallyoccurring oils such that the DHA to EPA ratio are within the scopedescribed herein. In some embodiments, the source of DHA may be agenetically modified plant or a genetically modified microorganismmanipulated to produce DHA.

In some embodiments, the composition of DHA is a microbial oil or isderived from a microbial oil. “Microbial oil” refers to those oilsnaturally produced by microorganisms. “Derived from” refers to amodification of the microbial oil, such as esters prepared from themicrobial oil; isolated or purified components of the microbial oil; orother processing of the microbial oil, such as concentration of the oil,to alter the percent weight of a component of the microbial oil.Exemplary microbes from which microbial oil may be obtained, include,among others, the microbial groups Stramenopiles, Thraustochytrids, andLabrinthulids. Stramenopiles includes microalgae and algae-likemicroorganisms, including the following groups of microorganisms:Hamatores, Proteromonads, Opalines, Develpayella, Diplophrys,Labrinthulids, Thraustochytrids, Biosecids, Oomycetes,Hypochytridiomycetes, Commation, Reticulosphaera, Pelagomonas,Pelagococcus, Ollicola, Aureococcus, Parmales, Diatoms, Xanthophytes,Phaeophytes (brown algae), Eustigmatophytes, Raphidophytes, Synurids,Axodines (including Rhizochromulinaales, Pedinellales, Dictyochales),Chrysomeridales, Sarcinochrysidales, Hydrurales, Hibberdiales, andChromulinales. The Thraustochytrids include the genera Schizochytrium(species include aggregatum, limnaceum, mangrovei, minutum, octosporum),Thraustochytrium (species include arudimentale, aureum, benthicola,globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum,roseum, striatum), Ulkenia (species include amoeboidea, kerguelensis,minuta, profunda, radiate, sailens, sarkariana, schizochytrops,visurgensis, yorkensis), Aplanochytrium (species include haliotidis,kerguelensis, profunda, stocchinoi), Japonochytrium (species includemarinum), Althornia (species include crouchii), and Elina (speciesinclude marisalba, sinorifica). The Labrinthulids include the generaLabyrinthula (species include algeriensis, coenocystis, chattonii,macrocystis, macrocystis atlantica, macrocystis macrocystis, marina,minuta, roscofensis, valkanovii, vitellina, vitellina pacifica,vitellina vitellina, zopfi), Labyrinthomyxa (species include marina),Labyrinthuloides (species include haliotidis, yorkensis), Diplophrys(species include archeri), Pyrrhosorus* (species include marinus),Sorodiplophrys* (species include stercorea), and Chlamydomyxa* (speciesinclude labyrinthuloides, montana) (*=there is no current generalconsensus on the exact taxonomic placement of these genera).

In some embodiments, the microbial oil source is oleaginousmicroorganisms, such as certain marine algae. As used herein,“oleaginous microorganisms” are defined as microorganisms capable ofaccumulating greater than 20% of the dry weight of their cells in theform of lipids. In some embodiments, the DHA is obtained or derived froma phototrophic or heterotrophic single cell organism or multicellularorganism, e.g., an algae. Thus, in some embodiments, the microbial oilis an algal oil. For example, the DHA may be obtained or derived from adiatom, e.g,. a marine dinoflagellates (algae), such as Crypthecodiniumsp., Thraustochytrium sp., Schizochytrium sp., or combinations thereof.Exemplary samples of C. cohnii, have been deposited with the AmericanType Culture Collection at Rockville, Md., and assigned the accessionnumbers 40750, 30021, 30334-30348, 3054130543, 30555-30557, 30571,30572, 30772-30775, 30812, 40750, 50050-50060, and 50297-50300.

As used herein, the term microorganism, or any specific type oforganism, includes wild strains, mutants or recombinant types. Organismswhich can produce an enhanced level of oil containing DHA are consideredto be within the scope of this invention. For example, cultivation ofdinoflagellates such as C. cohnii has been described previously. See,e.g., U.S. Pat. No. 5,492,938 and Henderson et al., Phytochemistry27:1679-1683 (1988). Also included are microorganisms designed toefficiently use more cost-effective substrates while producing the sameamount of DHA as the comparable wild-type strains.

Organisms useful in the production of DHA can also include any manner oftransgenic or other genetically modified organisms, such as agenetically modified plant or a genetically modified microorganismmanipulated to produce DHA. e.g., plants, grown either in culturefermentation or in crop plants, e.g., cereals such as maize, barley,wheat, rice, sorghum, pearl millet, corn, rye and oats; or beans,soybeans, peppers, lettuce, peas, Brassica species (e.g., cabbage,broccoli, cauliflower, brussel sprouts, rapeseed, and radish), carrot,beets, eggplant, spinach, cucumber, squash, melons, cantaloupe,sunflowers, safflower, canola, flax, peanut, mustard, rapeseed,chickpea, lentil, white clover, olive, palm, borage, evening primrose,linseed, and tobacco. In some embodiments, the DHA is derived from asoybean source, including wild type and genetically modified soybeansources.

In some embodiments, the DHA may be purified in the form of free fattyacids, fatty acid esters, phospholipids, triglycerides, diglycerides,monoglycerides or combinations thereof by any means known to those ofskill in the art. In some embodiments, the DHA comprises an ester. Theterra “ester” refers to the replacement of the hydrogen in thecarboxylic acid group of the DHA molecule with another substituent.Typical esters are known to those in the art, a discussion of which isprovided by Higuchi, T. and V. Stella in “Pro-drugs as Novel DeliverySystems,” Vol. 14, A.C.S. Symposium Series, Bioreversible Carriers inDrug Design, Ed. Edward B. Roche, American Pharmaceutical Association,Pergamon Press (1987), and Protective Groups in Organic Chemistry,McOmie ed., Plenum Press, New York (1973). In some embodiments, theester is an alkyl ester. Examples of more common esters include C1-C6esters, e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, or branchedvariations thereof, e.g., isopropyl, isobutyl, isopentyl, or t-butyl. Insome embodiments, the ester is a carboxylic acid protective ester group,esters with aralkyl (e.g., benzyl, phenethyl), esters with lower alkenyl(e.g., allyl, 2-butenyl), esters with lower-alkoxy-lower-alkyl (e.g.,methoxymethyl, 2-methoxyethyl, 2-ethoxyethyl), esters withlower-alkanoyloxy-lower-alkyl (e.g., acetoxymethyl, pivaloyloxymethyl,1-pivaloyloxyethyl), esters with lower-alkoxycarbonyl-lower-alkyl (e.g.,methoxycarbonylmethyl, isopropoxycarbonylmethyl), esters withcarboxy-lower alkyl (e.g., carboxymethyl), esters withlower-alkoxycarbonyloxy-lower-alkyl (e.g., 1-(ethoxycarbonyloxy)ethyl,1-(cyclohexyloxycarbonyloxy)ethyl), esters with carbamoyloxy-lower alkyl(e.g., carbamoyloxymethyl), and the like. In some embodiments, the addedsubstituent is a cyclic hydrocarbon group, e.g., C1-C6 cycloalkyl, orC1-C6 aryl ester. Other esters include nitrobenzyl, methoxybenzyl,benzhydryl, and trichloroethyl. In some embodiments, the estersubstituent is added to a DHA free acid molecule when the DHA is in apurified or semi-purified state. Alternatively, the DHA ester is formedupon conversion of a triglyceride to a ester. One of skill in the artcan appreciate that some non-esterified DHA molecules can be present inthe DHA compositions, e.g., DHA molecules that have not been esterified,or DHA triglyceride ester linkages that have been cleaved, e.g.,hydrolyzed. In some embodiments, the non-esterified DHA molecules or theDHA triglyceride molecules constitute less than 3% (mol/mol), about0.01% to about 2% (mol/mol), about 0.05% to about 1% (mol/mol), or about0.01% to about 0.5% (mol/mol) of the total DHA molecules. In someembodiments, the amount of ethyl ester of DHA in the compositions may beat least about 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt %.

In some embodiments, the DHA of the present invention is a triglyceride,diglyceride or monoglyceride. A “triglyceride” is a glyceride in whichthe glycerol is esterified with three fatty acid groups. Typicaltriglycerides are known to those in the art. In some embodiments, theDHA is in the form of a triglyceride or a diglyceride, wherein one ormore fatty acid groups other than DHA are present in the triglyceride ordiglyceride. In some embodiments, DHA is the only fatty acid group on atriglyceride or diglyceride molecule. In some embodiments, one or morefatty acid groups of a triglyceride have been hydrolyzed, or cleaved.

In some embodiments, the DHA of the present invention is in the form offree fatty acid. “Free fatty acid” refers to fatty acid compounds intheir acidic state, and salt derivatives thereof.

In some embodiments, the DHA may be purified in the form of free fattyacids, fatty acid esters, phospholipids, or triglycerides by any meansknown to those of skill in the art. Processes for the preparation ofvarious forms of DHA are described in, among others, U.S. Pub. No.2009/0023808 “Production and Purification of Esters of PolyunsaturatedFatty Acids” by Raman et al. and U.S. Pub. No. 2007/0032548“Polyunsaturated fatty acids for treatment of dementia andpre-dementia-related conditions” by Ellis, incorporated herein byreference. As used herein, “ester” refers to a molecule wherein thehydrogen in the carboxylic acid group of the DHA molecule has beenreplaced with another substituent. Examples of common esters includemethyl, ethyl, propyl, butyl, pentyl, t-butyl, benzyl, nitrobenzyl,methoxybenzyl, benzhydryl, and trichloroethyl. In some embodiments, theester is an alkyl ester, e.g., a methyl ester, ethyl ester or propylester. In some embodiments, the ester substituent is added to the DHAfree acid molecule when the DHA is in a purified or semi-purified state.Alternatively, the DHA ester is formed upon conversion of a triglycerideto a ester. In some embodiments, the amount of alkyl ester of DHA in thecompositions can be at least about 80, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or 99 wt %. In some embodiments, DHA alkyl esteris at least about 85 wt % of the total fatty acid content of thecomposition. In certain embodiments, the DHA alkylester is about 85 to96 wt % of the total fatty acid content. In some embodiments, thecomposition has a DHA alkyl ester content of about 85 to 96 wt % of thetotal fatty acid content, and an EPA content of about 0.1 wt % or lessof the total fatty acid content. In certain embodiments, the DHA alkylester is an ethyl ester. One of skill in the art can appreciate thatsome non-esterified DHA molecules may be present in the composition,e.g., DHA molecules that have not been esterified, or DHA ester linkagesthat have been cleaved, e.g., hydrolyzed. In some embodiments, thenonesterified DHA molecules constitute less than 3% (mol/mol), about 2%to about 0.01% (mol/mol), about 1% to about 0.05% (mol/mol), or about 5%to about 0.1% (mol/mol) of the total DHA molecules. Alternatively, insome embodiments, the DHA, may be purified in the free acid folin or ina salt form.

An exemplary method for producing a DHA ester may comprise: a) reactingthe composition comprising DHA in the presence of an alcohol and a baseto produce an ester of a polyunsaturated fatty acid from the DHA intriglycerides; and b) distilling the composition to recover a fractioncomprising the ester of the polyunsaturated fatty acid, optionallywherein the method further comprises: c) combining the fractioncomprising the ester of the polyunsaturated fatty acid with urea in amedium; d) cooling or concentrating the medium to form a urea-containingprecipitate and a liquid fraction; and e) separating the precipitatefrom the liquid fraction. In some embodiments, the purification processincludes starting with refined, bleached, and deodorized oil (RBD oil),then performing low temperature fractionation using acetone to provide aconcentrate. See U.S. application Ser. No. 12/163,555, incorporatedherein by reference. The concentrate may be obtained by base-catalyzedtransesterification, distillation, and silica refining to produce a DHAproduct.

As noted above, methods of determining purity levels of fatty acids areknown in the art, and may include, e.g., chromatographic methods suchas, e.g., HPLC silver ion chromatographic columns. Alternatively, puritylevels may be determined by gas chromatography, with or withoutconverting DHA to the corresponding alkyl ester. The percentage of fattyacids may also be determined using Fatty Acid Methyl Ester (FAME)analysis.

In some embodiments, the composition of DHA may include an additionallipid. As used herein, the term “lipid” includes phospholipids (PL);free fatty acids; esters of fatty acids; triacylglycerols (TAG);diacylglycerides; monoacylglycerides; phosphatides; waxes (esters ofalcohols and fatty acids); sterols and sterol esters; carotenoids;xanthophylls (e.g., oxycarotenoids); hydrocarbons; and other lipidsknown to one of ordinary skill in the art. The lipid can be chosen tohave minimal adverse health effects or minimally affect theeffectiveness of DHA when administered in combination with DHA.

In some embodiments, the composition of DHA may include an additionalunsaturated lipid. In some embodiments, the unsaturated lipid is apolyunsaturated lipid, such as another omega-3 fatty acid or an omega-6fatty acid. An exemplary omega-6 fatty acid that may be used in thecomposition is docosapentaenoic acid (DPA), including DPAn-6 or DPAn-3.

In some embodiments the dosage form comprises 0.1% to 20% of one or moreof the following fatty acids: (a) capric acid; (b) lauric acid; (c)myristic acid; (d) palmitic acid; (e) palmitoleic acid; (f) stearicacid; (g) oleic acid; (h) linoleic acid; (i) α-linolenic acid; j) DPAn-3 (22:5, n-3); (k) DPA n-6 (22:5, n-6); and (l) 4,7,10,13,16,19,22,25octacosaoctaenoic acid (C28:8). In some embodiments, the dosage foamcomprises 1% to 5% of one or more of the following fatty acids: (a)capric acid; (b) lauric acid; (c) myristic acid; (d) palmitic acid; (e)palmitoleic acid; (f) stearic acid; (g) oleic acid; (h) linoleic acid;(i) α-linolenic acid; (j) DPA n-3 (22:5, n-3); (k) DPA n-6 (22:5, n-6);and (l) 4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8). In someembodiments, the dosage form comprises less than 1% each of thefollowing fatty acids: (a) capric acid; (b) lauric acid; (c) myristicacid; (d) palmitic acid; (e) palmitoleic acid; (1) stearic acid; (g)oleic acid; (h) linoleic acid; (i) α-linolenic acid; (j)docosapentaenoic acid 22:5n-3, 22:5w3 (DPAn-3); (k) docosapentaenoicacid 22:5n-6, 22:5w6 (DPAn-6); and (l) 4,7,10,13,16,19,22,25octacosaoctaenoic acid (C28:8).

In some embodiments, the DHA composition may comprise DHASCO®. DHASCO®is an oil derived from Crypthecodinium cohnii containing high amounts ofdocosahexaenoic acid (DHA), and more specifically contains the followingapproximate exemplary amounts of these fatty acids, as a percentage ofthe total fatty acids: myristic acid (14:0) 10-20%; palmitic acid (16:0)10-20%; palmitoleic acid (16:1) 0-2%; stearic acid (18:0) 0-2%; oleicacid (18:1) 10-30%; linoleic acid (18:2) 0-5%; arachidic acid (20:0)0-1%; behenic acid (22:0) 0-1%; docosapentaenoic acid (22:5) 0-1%;docosahexanoic acid (22:6) (DHA) 40-45%; nervonic acid (24:1) 0-2%; andothers 0-3%. The composition of DHASCO is also described in U.S. Pat.No. 5,397,591 by Kyle et al., U.S. Pat. No. 5,407,957 by Kyle et al.,U.S. Pat. No. 5,492,938 by Kyle et al., and U.S. Pat. No. 5,711,983 byKyle et al.; the references of which are incorporated herein byreference. As will be understood by the skilled artisan, the content ofvarious components may vary because of variations on the manufacturingprocesses, with variations of DHA content being about 40 to 50 wt % ofthe total fatty acid content. In some embodiments, the DPAn-6 to EPAratio is about 6:1 to about 7:1 wt/wt, and the DHA to DPAn-6 ratio isabout 2:1 to about 3:1 wt/wt.

Alternatively, in some embodiments, the DHA composition may compriseLife's DHA™ (also formerly referenced as DHA™-S and DHASCO®-S), an oilderived from the Thraustochytrid, Schizochytrium sp., that contains ahigh amount of DHA and also contains docosapentaenoic acid (n-6)(DPAn-6). More specifically, DHA™-S contains the following approximateexemplary amounts of these fatty acids, as a percentage of total fattyacids: myristic acid (14:0) 8.71%; palmitic acid (16:0) 22.15%; stearicacid (18:0) 0.66%; linoleic acid (18:2) 0.46%; arachidonic acid (20:4)0.52%; eicosapentenoic acid (20:5, n-3) 1.36%; docosapentaenoic acid(22:5, n-6) (DPAn-6) 16.28%; docosahexaenoic acid (DHA) (22:6, n-3)41.14%; and others 8%. The characteristics of DHASCO®-S are alsodescribed in Ryan et al., Amer. J. Therapeutics 16:183-192(2009),incorporated herein by reference. As will be understood by the skilledartisan, the content of various components may vary because ofvariations on the manufacturing processes, with variations of DHAcontent being about 40 to 50 wt % of the total fatty acid content. DHAalkylesters are also described in U.S. application Ser. No. 12/572,263,filed Oct. 1, 2009, incorporated herein by reference. DHA alkylesterscan be prepared by techniques known in the art, such as U.S. Pat. No.6,395,778, incorporated herein by reference.

In some embodiments, the composition comprises DHA in the form of anethyl ester derived from Crypthecodinium cohnii, with the ester beingabout 89 wt % of the total fatty acid content of the composition. Morespecifically, the compositions contain the following exemplary amountsof the following fatty acid esters (i.e., ethyl esters) by weight:docosahexaenoic acid (22:6 w3) 89%; myristic acid (14:0) 0.1%; palmiticacid (16:0) 0.48%; palmitoleic acid (16:1 w7) 0.39%; oleic acid (18:1w9) 3.9%; docosapentaenoic acid (22:5 w3) 1.26%; octacosaoctaenoic acid(28:8 w3) 0.87%; tetracosaenoic acid (24:1 w9) 0.29%; and others 4.87%.EPA is not detectable by known methods of measuring EPA. As will beunderstood by the skilled artisan, the content of various components mayvary because of variations on the manufacturing processes, withvariations of DHA ethyl ester content being 85 to 96 wt % of the totalfatty acid content.

In some embodiments, the dosage form comprises, measured in weightpercent of the total free fatty acid content, about 35-65%, 40-55%,35-57%, or 57-65% DHA (22:6 n-3); about 0-2% capric acid (10:0); about0-6% lauric acid (12:0); about 10-20% myristic acid (14:0); about 5-15%palmitic acid (16:0); about 0-5% palmitoleic acid (16:1); about 0-2%stearic acid (18:0); about 5-20% or 5-25% oleic acid (18:1); about 0-2%linoleic acid (18:2); and about 0-2% nervonic acid (24:1n-9). In certainembodiments, such an oil is from a microorganism of the genusThraustochytrium. In other embodiments, the free fatty acid content isless than 0.4%. In some embodiments, the dosage foam comprises, measuredin weight percent of total free fatty acid content, about 35-45% DHA(22:6n-3); about 0-2% lauric acid (12:0); about 5-10% myristic acid(14:0); about 5-20% palmitic acid (16:0); about 0-5% palmitoleic acid(16:1); about 0-5% stearic acid (18:0); about 0-5% vaccenic acid oroleic acid (18:1 n-7 and n-9, respectively); about 0-2% linoleic acid(18:2, n-6); about 0-5% stearidonic acid (18:4 n-3); about 0-10% 20:4n-3, n-5, or n-6; about 0-2% adrenic acid 22:4 n-6; about 0-5% DPAn-3(22:5); about 10-25% DPAn-6 (22:5); and 0-2% 24:0. In some embodiments,such an oil is from a microorganism of the genus Schizochytrium.

An exemplary DHA (triglyceride) containing oil derived fromSchizochytrium sp. is characterized by the specified amount ofcomponents listed in Table 4, where “Max” refers to the amount of thecomponent that can be present up to the specified amount.

TABLE 4 FATTY ACIDS CONCENTRATION (WT/WT) 14:0 6.0%-12.0% 16:0  18%-28%18:0 MAX 2% 18:1 MAX 8% 18:2 MAX 2% 20:4 ARA MAX 2% 20:5 (N-3) EPA MAX3% 22:5 (N-6) DPA  12%-18% 22:6 (N-3) DHA MIN 35% OTHERS MAX 10%ELEMENTAL COMPOSITION ARSENIC MAX 0.2 PPM COPPER MAX 0.05 PPM IRON MAX0.2 PPM LEAD MAX 0.1 PPM MERCURY MAX 0.04 PPM CHEMICAL CHARACTERISTICSPEROXIDE VALUE MAX 5 MEQ/KG FREE FATTY ACID MAX 0.25% MOISTURE ANDVOLATILES MAX 0.05% UNSAPONIFIABLE MATTER MAX 4.5% TRANS FATTY ACIDS MAX1%

In some embodiments, the DHA-containing compositions can comprise atleast about 40 wt % DHA and at least about 0.1 wt % of4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8). In someembodiments, the compositions comprise at least about 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65 wt % DHA, optionally in triglyceride faun, asa percentage of total fatty acids. In other embodiments, thecompositions comprise at least about 90, 91, 92, 93, 94, 95, 96, 97, 98,or 99 wt % of DHA, optionally in ethyl ester foim, as a percentage oftotal fatty acids. In certain embodiments, the amount of C28:8 in thecompositions may be at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 wt %. The C28:8 may be present inany form, including triglyceride or ester form. For example, the C28:8may be present in ethyl ester form. In certain embodiments, thecompositions comprise all three of DHA, C28:8 and DPAn-3 in theconcentration ranges specified above.

The present invention also provides compositions comprising at leastabout 40 wt % DHA and at least about 0.1 wt % of DPAn-3. In someembodiments, the compositions comprise at least about 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65 wt % DHA, optionally in triglyceride form, asa percentage of total fatty acids. In other embodiments, thecompositions comprise at least about 90, 91, 92, 93, 94, 95, 96, 97, 98,or 99 wt % of DHA, optionally in ethyl ester form, as a percentage oftotal fatty acids. In certain embodiments, the amount of DPAn-3 in thecompositions may be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, or 1.0 wt % of DPAn-3. The DPAn-3 may be present intriglyceride or ester form. For example, the DPAn-3 may be present inethyl ester form.

In certain embodiments, the compositions comprise all three of the DHA,C28:8 and DPAn-3 in the concentration ranges specified above.

In further embodiments, the compositions may comprise less than about1.0, 0.9, 0.8. 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 wt % EPA in additionto the DHA and C28:8. In one embodiment, the compositions may compriseless than about 0.25 wt % EPA. The EPA may be present in any form,including triglyceride or ester form. In some embodiments, thecompositions may comprise 0 wt % EPA.

The present invention also provides compositions comprising at leastabout 90 wt % of DHA and at least one additional fatty acid or aderivative thereof. In some embodiments, the amount of DHA in thecompositions may be at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99wt %. In certain embodiments, the additional fatty acid may have aboiling point of about 150-170° C. at a pressure of 0.8 mm Hg.

An exemplary DHA-containing oil derived from the algal oil ofCrypthecodinium Cohnii, wherein the DHA comprises an ethyl ester, can becharacterized by the specified amount of components listed in Table 5,where “Max” refers to the amount of the component that can be present upto the specified amount.

TABLE 5 DHA CONTENT (MG/G) 855-945 FATTY ACID CONTENT: % OF TOTAL EEEICOSAPENTAENOIC ACID (20:5.3) ND MYRISTIC ACID (14:0) 0.1% PALMITICACID (16:0) 0.5% PALMITOLEIC ACID (16:1.7) 0.4% STEARIC ACID (18:0) NDOLEIC ACID (18:1.9)   4% LINOLEIC ACID (18:2.6) ND DOCOSAPENTAENOIC ACID(22:5.3) 1.3% OCTACOSAOCTAENOIC ACID (28:8.3) 0.9% TETRACOSAENOIC ACID(24:1.9) 0.3% OTHERS 1.1% ELEMENTAL COMPOSITION ARSENIC MAX 0.5 PPMCOPPER MAX 0.1 PPM IRON MAX 0.5 PPM LEAD MAX 0.2 PPM MERCURY MAX 0.04PPM CHEMICAL CHARACTERISTICS PEROXIDE VALUE MAX 10.0 MEQ/KG ND = NOTDETECTABLE

In some embodiments, an oil is characterized by one or more thefollowing fatty acids (or esters thereof), expressed as wt % of thetotal fatty acid content. The embodiments provided herein may furthercomprise about 12% or less, or about 6% to about 12% (wt/wt) of myristicacid (C14:0). The embodiments provided herein may farther comprise about28% or less, or about 18 to about 28% (wt/wt) of palmitic acid (C16:0).The embodiments provided herein ma.y further comprise about 2% or less(wt/wt) of stearic acid (C18:0). The embodiments provided herein mayfurther comprise about 8% or less of (wt/wt) oleic acid (C18:1 n-9). Theembodiments provided herein may further comprise about 2% or less(wt/wt) of linoleic acid (C18:2). The embodiments provided herein mayfurther comprise about 2% or less (wt/wt) of arachidonic acid (C20:4).The embodiments provided herein may further comprise about 3% or less(wt/wt) of eicosapentaenoic acid (C20:5). The embodiments providedherein may further comprise about 18% or less, or about 12% to about 18%(wt/wt) of decosapentaenoic acid (22:5n-6). The embodiments providedherein may further comprise about 10% or less (wt/wt) of other fattyacids. In some of these embodiments, the ratio of wt % of DHA to wt % ofDPAn-6 is about 2.5 to about 2.7. An oil with the precedingcharacteristics may comprise Life's DHA™ (also formerly referenced asDHA™-S and DHASCO®-S), Martek Biosciences, Columbia, Md.), an oilderived from the Thraustochytrid, Schizochytrium sp., that contains ahigh amount of DHA and also contains decosapentaenoic acid (n-6)(DPAn-6).

The present invention further includes compositions comprising at leastabout 70 wt % DHA and at least about 15, 20, or 25 wt % DPAn-6.

Compositions of the present invention also include compositions thatcomprise at least about 90 wt % of a combination of DPAn-6 and DHA. Incertain embodiments, the compositions may comprise at least about 91,92, 93, 94, 95, 96, 97, 98, or 99 wt % of a combination of DPAn-6 andDHA. In some embodiments, the compositions may comprise at least about10 wt % DHA and at least about 10 wt % DPAn-6. In other embodiments, thecompositions may comprise at least about 15 or 20 wt % DHA and at leastabout 15 or 20 wt % DPAn-6.

The present invention also provides compositions comprising at leastabout 90 wt % of a combination of DPAn-6 and DHA, and at least oneadditional fatty acid or a derivative, such as an ester, thereof Incertain embodiments, the compositions may comprise at least about 91,92, 93, 94, 95, 96, 97, 98, or 99 wt % of a combination of DPAn-6 and.DHA. In some embodiments, the additional fatty acid may have a boilingpoint of about 150-170° C. at a pressure of 0.8 mm Hg.

The DHA/DPAn-6 compositions described above may further comprise lessthan about 4% of a saturated fatty acid or an ester thereof In certainembodiments, the compositions may comprise less than about 3.5%, 3.0%,2.5%, 2.0%, 1.5%, 1.0% or 0,5% of a saturated fatty acid or a derivativethereof.

The DHA in an oil may be in the form of a DHA ester, preferably an alkylester, such as a methyl ester, ethyl ester, propyl ester, orcombinations thereof, prepared from an algal oil derived from theThraustochytrid, Schizochytrium sp. An exemplary DHA (ethyl esters)containing oil derived from Schizochytrium sp. is characterized by thespecified amount of components listed in Table 4 of WO 2009/006317,incorporated by reference herein. In some of these embodiments, an oilcomprises DHA greater than about 57% (wt/wt), particularly greater thanabout 70% (wt/wt) of the total fatty acid content of the oil or unitdose. In some of these embodiments, the ratio of wt % of DHA to wt % ofDPAn-6 is about 2.5 to about 2.7.

In some embodiments, the composition or oil is characterized by one ormore the following fatty acids (or esters thereof, particularly ethylesters), expressed as wt % of the total fatty acid content. Theembodiments provided herein may further comprise about 0.5% or less(wt/wt) of lauric acid (C12:0). The embodiments provided herein mayfurther comprise about 2% or less (wt/wt) of myristic acid (C14:0). Theembodiments provided herein may further comprise about 0.5% or less(wt/wt) of myristoleic acid (C14:1). The embodiments provided herein mayfurther comprise about 1% or less of palmitic acid (C16:0). Theembodiments provided herein may further comprise about 1% or less(wt/wt) of linoleic acid (C18:2) (n-6). The embodiments provided hereinmay further comprise about 3% or less (wt/wt) of dihomo gamma linolenicacid (C20:3) (n-6). The embodiments provided herein may further compriseabout 0.5% or less (wt/wt) of eicosatrienoic (C20:3) (n-3). Theembodiments provided herein may further comprise about 1% or less(wt/wt) of arachidonic acid (C20:4). The embodiments provided herein mayfurther comprise about 3% or less (wt/wt) of eicosapentaenoic acid(C20:5) (n-3). The embodiments provided herein may further compriseabout 3% or less (wt/wt) of docosatrienoic acid (22:3). The embodimentsprovided herein may further comprise about 27% or less (wt/wt) ofdecosapentaenoic acid (22:5) (n-6). The embodiments provided herein mayfurther comprise about 10% or less (wt/wt) of other components. In someof these embodiments, the ratio of wt % of DHA to wt % of DPAn-6 isabout 2.5 to about 2.7. An oil with the preceding characteristics maycomprise ethyl ester oil derived from the oil of Thraustochytrid,Schizochytrium sp.

In some embodiments, another exemplary DHA (free fatty acid) containingoil is characterized by the specified amount of components (as ethylesters) listed in Table 6, where “Max” refers to the amount of thecomponent that can be present up to the specified amount.

TABLE 6 FATTY ACIDS CONCENTRATION (WT/WT) C12:0 MAX 0.5% C14:0 MAX 2%C14:1 MAX 0.5% C16:0 MAX 1% C18:2 N-6 MAX 1% C20:3 (N-6) MAX 3% C20:3(N-3) MAX 0.5% C20:4 ARA MAX 1% C20:5 (N-3) EPA MAX 3% C22:3 MAX 3%C22:5 (N-6) DPA MAX 27% C22:6 (N-3) DHA MIN 57% % ADDITIONAL COMPONENTSMAX 8%

In some embodiments, another exemplary DHA (free fatty acid) containingoil is characterized by the specified amount of components listed inTable 7:

TABLE 7 FATTY ACIDS CONCENTRATION (WT/WT) 10:0 MAX 0.5% 12:0 MAX 0.5%14:0 MAX 0.5% 14:1 MAX 0.5% 16:0 MAX 0.5% 16:1 MAX 0.5% 18:1 (N-9) MAX0.5% 20:5 (N-3) EPA MAX 0.5% 22:5 (N-3) DPA MAX 1% 22:6 (N-3) DHA MIN95% 28:8 MAX 1.5% CHEMICAL CHARACTERISTICS DOCOSAHEXAENOIC ACID 946 MG/GDOCOSAHEXAENOIC ACID 98% FREE FATTY ACIDS 93% TRANS FATTY ACIDS <1%

In some embodiments, the saturated fatty acid or an ester thereof maycontain less than 20 carbons, such as, for example, a saturated fattyacid or an ester thereof that contains 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9 or 8 carbons. In certain embodiments, the saturated fatty acidor ester thereof may contain 14 or 16 carbons.

In some embodiments, the composition of DHA may further comprise vitaminE.

Compounds of the vitamin E group are fat-soluble vitamins withantioxidant properties and includes eight related α-, β-, γ-, andδ-tocopherols and the corresponding four tocotrienols. In someembodiments, the vitamin E in the composition is a tocopherol. In someembodiments, the tocopherol is selected from α-, β-, γ-, andδ-tocopherols, or combinations thereof.

Subject as used herein refers to a human subject at risk for sufferingtraumatic brain injury. The patient can be at risk, now or at some timein the future, of suffering traumatic brain injury, including mild,moderate and severe forms of closed brain injury or penetrating braininjury.

In the embodiments herein, the compositions of DHA are administered inan amount effective to reduce the risk of pathological effects oftraumatic brain injury. As used herein, “reduce the risk of pathologicaleffects” refer to prophylactic therapeutic treatment, wherein the objectis to treat or ameliorate the undesired physiological condition ordisorder, or obtain beneficial or desired clinical results when thesubject is afflicted with the condition or disorder. For purposesherein, beneficial or desired clinical results include, but are notlimited to, reducing or alleviating the symptoms associated withtraumatic brain injury; diminishment of the extent of the conditionassociated with traumatic brain injury; reducing or alleviating thecondition or disorder of traumatic brain injury, whether detectable orundetectable; or enhancement or improvement of the condition or disorderassociated with traumatic brain injury. Prophylactic treatment alsoincludes prolonging survival as compared to expected survival if notreceiving treatment.

For the purposes herein, the composition of DHA is administered in aprophylactically effective amount to reduce the risk of sufferingpathological effects of traumatic brain injury. As used herein, a“prophylactically effective amount” refers to an amount of DHA effectivein achieving a desired therapeutic response in reducing the pathologicaleffects of traumatic brain injury. A prophylactically effective amountof DHA may vary according to factors such as age, sex, and weight of theindividual. Administration of a prophylactically effective amount of DHAmay be achieved using various regimens, including variations infrequency and time period, sufficient to provide a therapeutic benefitto the subject who suffers traumatic brain injury. In some embodiments,administration of the DHA is daily on consecutive days, oralternatively, the dosage form is administered every other day(bi-daily). Administration may occur on one or more days.

In some embodiments, a prophylactically effective amount of DHA isadministered to the subject for a sufficient time period prior to ananticipated engagement in an activity associated with a risk oftraumatic brain injury. In some embodiments, a prophylacticallyeffective amount of DHA is administered for at least about 28 days priorto the anticipated engagement in the activity that is associated with arisk of traumatic brain injury. In some embodiments, a prophylacticallyeffective amount of DHA is administered for at least about 6 weeks priorto the anticipated engagement in the activity that is associated with arisk of traumatic brain injury. In some embodiments, a prophylacticallyeffective amount of DHA is administered for at least about two monthsprior to the anticipated engagement in the activity that is associatedwith a risk of traumatic brain injury. In some embodiments, aprophylactically effective amount of DHA is administered to the subjectfor at least about 6 weeks to about 6 months, at least about 2 to about6 months, or at least about 4 months to about 6 months prior to theanticipated engagement in the activity that is associated with a risk oftraumatic brain injury. In some embodiments, a prophylacticallyeffective amount of DHA is administered to the subject for at leastabout 2 to about 4 months prior to the anticipated engagement in theactivity that is associated with a risk of traumatic brain injury.

As used herein, “daily dose,” “daily dosage level,” “daily dosageamount” or “per day dosage” refer to the total amount of DHA (e.g., inthe form of free fatty acids, alkyl esters, or triglycerides)administered per day (about 24 hour period). For example, administrationof DHA to a subject at a dose of 2 g per day means that the subjectreceives a total of 2 g of DHA on a daily basis, whether the DHA isadministered as a single dosage form comprising 2 g DHA, oralternatively, four dosage forms comprising 500 mg DHA each (for a totalof 2 g DHA). The composition of DHA may be taken in a single applicationor multiple applications per day. For example, if four capsules aretaken daily, each capsule comprising 500 mg DHA, then all four capsulescould be taken once per day, or 2 capsules could be taken twice per day,or 1 capsule could be taken every 6 hours. In some embodiments, thecomposition comprising DHA is administered at least once per day (e.g.,single dosage form daily) or at least twice per day (e.g., in two ormore dosage forms daily). In some embodiments, the DHA is administeredat least two times weekly.

In some embodiments, the DHA is administered in an amount of from about4 mg/kg body weight/day to about 85 mg/kg body weight/day. In someembodiments, the DHA is administered in an amount of from about 4 mg/kgbody weight/day to about 60 mg/kg body weight/day; from about 5 mg/kgbody weight/day to about 60 mg/kg body weight/day, from about 10 mg/kgbody weight/day to about 60 mg/kg body weight/day, from about 20 mg/kgbody weight/day to about 60 mg/kg body weight/day; from about 10 mg/kgbody weight/day to about 40 mg/kg body weight/day; or from about 20mg/kg body weight/day to about 40 mg/kg body weight/day. In someembodiments, the DHA is administered in an amount of about 40 mg/kg bodyweight/day.

In some embodiments, the DHA is administered in an amount of from about300 mg to about 6 g per day; from about 0.5 g per day to about 6 g perday; from about 1 g per day to 6 g per day; or from about 2 g per day to6 g DHA per day. In some embodiments, the DHA is administered in anamount from about 300 mg to about 5 g per day, from about 0.5 g per dayto about 5 g per day; from about 1 g per day to about 5 g per day, orfrom 2 g per day to about 5 g DHA per day. In some embodiments, the DHAis administered in an amount from about 300 mg to about 4 g per day,from about 0.5 g per day to about 4 g per day, from about 1 g per day toabout 4 g per day, or from about 2 g per day to about 4 g DHA per day.

In some embodiments, the DHA is administered in an amount of from about1.5 mg per kg body weight per day to about 125 mg per kg body weight perday. In some embodiments, the DHA is administered in an amount of fromabout 150 mg to about 10 g per day; from about 0.5 g per day to about 5g per day; or from about 1 g per day to about 5 g per day.

In some embodiments, the daily amount of DHA administered comprisesabout 200 mg, 400 mg, 450 mg, 500 mg, 520 mg, 540 mg, 600 mg, 700 mg,800 mg, 900 mg, 1 g, 1.5 g, g, 2.0 g, 2.5 g, 2.7 g, 3.0 g, 3.2g, 3.3g,3.4 g, 3.5 g, 3.6 g, 3.7 g, 3.8 g, 3.9 g, 4.0 g, 4.5 g, 5.0g, 6.0 g,6.5 g, 7 g, 8 g, 9 g, or 10 g DHA per day. In some embodiments, the DHAis administered in an amount of at least about 1 g per day.

In some embodiments, the daily dose of DHA administered to a humansubject ranges from about 860 mg up to about 6 g, particularly fromabout 1.7 g up to about 6 g, from about 2.6 g up to about 6 g,particularly from about 3.4 g up to about 6 g, particularly from about4.3 g to about 6 g and more particularly from about 5.1 g to about 6 g.In some embodiments the daily dose of DHA administered to a humansubject ranges from about 860 mg up to about 4 g, particularly fromabout 1.7 g up to about 4 g, from about 2.6 g up to about 4 g, and moreparticularly from about 3.4 g up to about 4 g. In some embodiment thedaily dose of DHA administered to a human subject ranges from about 860mg up to about 1 g, particularly from about 860 mg up to about 950 mg.In some embodiments, the daily dose of DHA administered ranges fromabout 1.7 g up to about 2 g, particularly from about 1.7 g up to about1.8 g. In some embodiments, the daily dose of DHA administered to ahuman subject ranges from about 2.6 g up to about 3 g, particularly fromabout 2.6 g up to about 2.8 g. In some embodiments, the daily dose ofDHA administered to a human subject is from about 3.4 g up to about 4 g,particularly from about 3.4 g up to about 3.8 g. In some embodiments,the daily dose of DHA administered to a human subject is from about 4.3to about 5 g, particularly from 4.3 g to about 4.8 g. In someembodiments, the daily dose of DHA administered to a human subject isfrom about 5.1 to about 6 g, particularly from about 5.1 to about 5.7 g.

In some embodiments, the daily dose is provided as a unit dose.

In some embodiments, the amount of DHA administered comprises about 300mg, 400 mg, 450 mg, 500 mg, 520 mg, 540 mg, 600 mg, 700 mg, 800 mg, 900mg, 1 g, 1.5 g, 1.8 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 5.0 g, or 6.0g of DHA per day. In some embodiments, the DHA is administered in anamount of about 2 g per day.

In some embodiments, various amounts of DHA may be in a dosage form. Insome embodiments, the dosage form comprises less than about 5 g of DHA,about 100 mg to about 3.8 g DHA, about 200 mg to about 3.6 g of DHA,about 500 mg to about 4.0 g DHA, or about 1 g to about 2.0 g DHA. Insome embodiments, the dosage form comprises less than about 4 g of DHA,about 200 mg to about 3.9 g DHA, about 500 mg to about 3.7 g of DHA,about 750 mg to about 3.5 g DHA, or about 1 g to about 2 g DHA. In someembodiments, the dosage form of DHA is less than about 3.8 g DHA, about900 mg to about 3.6 g DHA, or about 1.8 g to about 2.7 g of DHA. In someembodiments, the dosage foam of DHA comprises about 200 mg, 400 mg, 450mg, 500 mg, 900 mg, 1 g, 1.5 g, 1.8 g, 2.0 g, 2.5 g, 2.7 g, 3.0 g, 3.2g, 3.3 g,3.4 g, 3.5 g, 3.6 g, 3.7 g, 3.8 g, 3.9 g, 4.0 g, 4.5 g, 5.0 g,6.0 g, 6.5 g, 7 g, 8 g, 9 g, or 10 g DHA.

Administration of the DHA may be achieved using various regimens. Forexample, in some embodiments, administration of the DHA is daily onconsecutive days, or alternatively, the dosage form is administeredevery other day (bi-daily). Administration may occur on one or moredays. For example, in some embodiments the DHA is administered daily forthe duration of the subject's lifetime, or from 1 year to 20 years or 5years to 10 years. In some embodiments, administration of the DHA dosageform occurs for 7, 14, 21, or 28 days. In some embodiments, the DHA isadministered for at least 6 months, for at least 1 yr, for at least 1.5yrs., for at least 2 yrs., or for at least 5 yrs. In some embodiments,administration of the DHA occurs until a symptom of dementia or AD,e.g., loss of cognitive ability, is halted or reduced, the target beingdetermined by a medical professional.

In some embodiments, the DHA is administered continuously. The term“continuous” or “consecutive,” as used herein in reference to“administration,” means that the frequency of administration is at leastonce daily. Note, however, that the frequency of administration can begreater than once daily and still be “continuous” or “consecutive,”e.g., twice or even three or four times daily, as long as the dosagelevels as specified herein are achieved.

In some embodiments, the amount of DHA administered is an amount thatresults in maximal amount of DHA in brain phospholipids that isachievable by administration of the DHA containing composition. In someembodiments, the DHA composition is administered to achieve an amount ofDHA in the brain phospholipid fraction of at least about 15 wt % oftotal brain phospholipid. In some embodiments, the DHA composition isadministered to achieve an amount of DHA in the brain phospholipidfraction of about 15-22 wt % of total brain phospholipid content. Insome embodiments, the DHA composition is administered to achieve anamount of DHA in the brain phospholipid fraction of about 18-20 wt % oftotal brain phospholipid content. The amount of DHA administered and theduration of administration required to achieve the indicated DHA levelsin the brain phospholipid fraction can be determined by those skilled inthe art. In some embodiments, the composition comprising DHA isadministered to provide a DHA dose of about 2 g per day to 4 g per dayto achieve the maximal amount of DHA present in the brain phospholipids.

The effectiveness of the treatment can be assessed using methodsgenerally accepted in the art for determining the severity of traumaticbrain injury, such as brain imaging techniques, including computerassisted tomography (CAT) scans, which allow visualization of fracturesand evidence of bleeding in the brain (hemorrhage), large blood clots(hematomas), bruised brain tissue (contusions) and brain tissueswelling; and magnetic resonance imaging (MRI), including Susceptibilityweighted images (SWI), a sensitive method for detecting smallhemorrhages in the brain, and Diffusion tensor imaging (DTI), whichconsists of a minimum of six scans with diffusion gradients placed in anorthogonal manner. In some embodiments, traumatic brain injury can beassessed by measuring intracranial pressure, which can occur by swellingof the brain.

Since neurobehavioral, particularly cognitive related, problems are amajor effect of traumatic brain injury, various methods used to assesscognitive function can be used to assess the effectiveness ofprophylactic treatment. Such assessments include, among others, thefollowing. Clinical Dementia Rating Scale (CDR), a dementia staginginstrument that classifies cognitive impairment along a continuum fromnormal aging to mild cognitive impairment to all stages of dementiaseverity; Folstein Mini-Mental State Exam (MMSE), a commonly usedmeasure of orientation and gross cognitive functioning used byphysicians and healthcare providers to screen for cognitive decline; andAlzheimer's Disease Assessment Scale-Cognitive (ADAS-C), a test commonlyused in detection of dementia and MCI with repeated measures designs.

Additional methods for assessing cognitive impairment from traumaticbrain injury can include, among others, various neuropsychological test,such as the following: Wechsler Test of Adult Reading (WTAR), which is ameasure of word pronunciation and is a reliable predictor of pre-morbidgeneral intellectual function; Wechsler Adult Intelligence Scale-3(WAIS-3)—Kaufman tetrad short form, which is used to measure generalintellectual functioning; Repeatable Battery for the Assessment ofNeuropsychological Status (RBANS), a comprehensive but relatively rapid,standardized measure of neurocognitive functioning in multiple domains,including memory, attention, language, and visuospatial/constructionalfunctions; Trailmaking Test Part A (Trails A), a widely-used measure ofcognitive processing and visuomotor speed, and with Part B, alsopreviously employed in studies of MCI; Trailmaking Test Part B (TrailsB), a more complex measure of cognitive processing with executivedemands related to mental flexibility and working memory; ControlledOral Word Association Test (COWAT), a well-known measure ofphonemically-controlled verbal fluency, sensitive to cognitive slowingand impairments of executive functioning, which is routinely employed indementia assessment and MCI studies; Boston Naming Test (BNT), a visualconfrontation naming measure utilized to detect anomia or word-findingdifficulties, which are common hallmarks of cognitive decline in elderlypopulations with mild cognitive impairment or early dementia; AutomatedNeuropsychological Assessment Metrics (ANAM), a computerized testdesigned to assess several cognitive domains known to be sensitive tochange following concussion, including attention and concentration,reaction time, working memory, new learning and memory, and speed ofinformation processing; and SF-36, which measures eight domains ofhealth, including physical functioning, role limitations due to physicalhealth, bodily pain, general health perceptions, vitality, socialfunctioning, role limitations due to emotional problems, and mentalhealth.

In some embodiments, the subject is a human, and as administered acomposition comprising DHA as described herein in a range of about 5mg/kg/day to about 40 mg/kg/day for an extended period of time, e.g., 1month to 3 months, prior to the traumatic brain injuring event. In someembodiments, the administration of compositions comprising DHA prior tothe traumatic brain injuring event mitigates the adverse effects fromneuro-inflammation and supports normal brain function following a mildto moderate/severe brain injury, particularly in the absence ofpenetrating wounds and “excessive” structural damage to the brain. Insome embodiments, the administration of compositions comprising DHAprior to the traumatic brain injuring event supports normal energymetabolism in neurons following a brain injury. In some embodiments, theadministration of compositions comprising DHA prior to the traumaticbrain injuring event maintains and/or improves structural integrity andfunction of neurons and neuronal axons following mild to moderate braininjury. In some embodiments, the administration of compositionscomprising DHA prior to the traumatic brain injuring event supportsneuron survival and function following a mild to moderate brain injury.In some embodiments, the administration of compositions comprising DHAprior to the traumatic brain injuring event supports white-matterintegrity and optimal (e.g., normal) neurotransmission. In someembodiments, the administration of compositions comprising DHA prior tothe traumatic brain injuring event facilitates normal cognitive functionpost-injury. In some embodiments, the administration of compositionscomprising DHA prior to the traumatic brain injuring event supportnormal memory and learning functions post-injury.

The composition of DHA may be formulated in pharmaceutically acceptabledosage forms. “Pharmaceutically acceptable” refers to compositions thatare, within the scope of sound medical judgment, suitable for contactwith the tissues of human beings and animals without excessive toxicityor other complications commensurate with a reasonable benefit/riskratio. In some embodiments, the compounds (e.g., DHA), compositions, anddosage forms of the present invention are pharmaceutically acceptable.These dosage forms may include, but are not limited to, tablets(including chewable tablets, quick dissolve tablets, effervescenttablets, multi-layer and bi-layer tablets), capsules (including soft andhard gelatin capsules), caplets, cachets, lozenges (including chewablelozenges), beads, pellets, emulsions, liquid, pills, gel caps, elixirs,powders (including reconstitutable powders), granules, and dispersiblegranules; and parenteral dosage forms which include, but are not limitedto, solutions, suspensions, emulsions, particles, microparticles, coatedparticles, and dry powder comprising an effective amount of the DHA asprovided in this disclosure. Various substances are known in the art tocoat particles, including cellulose derivatives, e.g., microcrystallinecellulose, methyl cellulose, carboxymethyl cellulose; polyalkyleneglycol derivatives, e.g., polyethylene glycol; talc, starch,methacrylates, etc. In some embodiments, the dosage form is a capsule,wherein the capsule is filled with a solution, suspension, or emulsioncomprising the DHA. It is also known in the art that the activeingredients may be contained in such formulations with pharmaceuticallyacceptable excipients such as diluents, fillers, disintegrants, binders,lubricants, surfactants, hydrophobic vehicles, water soluble vehicles,emulsifiers, buffers, humectants, moisturizers, solubilizers,preservatives, flavorants, taste-masking agents, sweeteners, and thelike. Suitable excipients may include, e.g., vegetable oils (e.g., corn,soy, safflower, sunflower, or canola oil). In some embodiments, thepreservative may be an antioxidant, e.g., sodium sulfite, potassiumsulfite, metabisulfite, bisulfites, thiosulfates, thioglycerol,thiosorbitol, cysteine hydrochloride, tocopherol, and combinationsthereof. The means and methods for administration are known in the artand an artisan can refer to various pharmacologic references forguidance. For example, “Modern Pharmaceutics,” Banker & Rhodes, InformaHealthcare, 4th ed. (2002); and “Goodman & Gilman's The PharmaceuticalBasis o/Therapeutics,” McGraw-Hill, New York, 10th ed. (2001) can beconsulted.

In some embodiments, specifically excluded from the DHA-containingcompositions for administration in the treatment of traumatic braininjury is a complex of DHA and albumin, as described in U.S. Pub. No.2006/0094654, and Belayev et al., Stroke 36:118-23 (2005),electronically published Nov. 29, 2004.

Administration of DHA may be by oral or parenteral routes (e.g.,subcutaneous, intravenous (bolus or infusion), intramuscular, orintraperitoneal). In some embodiments, combinations of different routesof administration can be used. When administered by different routes,the administration can be done concurrently or sequentially. Forexample, a composition of DHA can be administered orally for chronicadministration (e.g., weeks, months before engaging in the activity withrisk for traumatic brain injury) and then parenterally to the subjectbefore engaging in an activity with risk for traumatic brain injury.Alternatively, the compositions of DHA can be administered concurrentlythrough different routes, for example parenteral and oral. The dosagefont's for these modes of administration may include conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.

In some embodiments, the route of administration is by oraladministration. The DHA composition can be administered to subjects inthe form of nutritional supplements, foods, pharmaceutical formulations,or beverages, particularly foods, beverages, or nutritional supplements,more particularly, foods and beverages, more particularly foods. Apreferred type of food is a medical food (e.g., a food which is in aformulation to be consumed or administered externally under thesupervision of a physician and which is intended for the specificdietary management of a disease or condition for which distinctivenutritional requirements, based on recognized scientific principles, areestablished by medical evaluation.).

In some embodiments, the dosage form is a pharmaceutical dosage form.“Pharmaceutically acceptable” refers to compositions that are, withinthe scope of sound medical judgment, suitable for contact with thetissues of human beings and animals without excessive toxicity or othercomplications commensurate with a reasonable benefit/risk ratio. In someembodiments, the compounds (e.g., DHA), compositions, and dosage formsof the present invention are pharmaceutically acceptable.

The DHA can be formulated in a dosage form. These dosage forms caninclude, but are not limited to, tablets, capsules, cachets, pellets,pills, gelatin capsules, powders, and granules; and parenteral dosageforms which include, but are not limited to, solutions, suspensions,emulsions, coated particles, and dry powder comprising an effectiveamount of the DHA as taught in this invention. In some embodiments, thedosage form can be inserted or mixed into a food substance. Varioussubstances are known in the art to coat particles, including cellulosederivatives, e.g., microcrystalline cellulose, methyl cellulose,carboxymethyl cellulose; polyalkylene glycol derivatives, e.g.,polyethylene glycol; talc, starch, methacrylates, etc. In someembodiments, the dosage form is a capsule, wherein the capsule is filledwith a solution, suspension, or emulsion comprising the DHA. It is alsoknown in the art that the active ingredients can be contained in suchformulations with pharmaceutically acceptable excipients such asdiluents, fillers, disintegrants, binders, lubricants, surfactants,hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers,humectants, moisturizers, solubilizers, preservatives, flavorants,taste-masking agents, sweeteners, and the like. Suitable excipients caninclude, e.g., vegetable oils (e.g., corn, soy, safflower, sunflower, orcanola oil). In some embodiments, the preservative can be anantioxidant, e.g., sodium sulfite, potassium sulfite, metabisulfite,bisulfites, thiosulfates, thioglycerol, thiosorbitol, cysteinehydrochloride, .-tocopherol, and combinations thereof The means andmethods for administration are known in the art and an artisan can referto various pharmacologic references for guidance. For example, “ModernPharmaceutics,” Banker & Rhodes, Informa Healthcare, 4th ed. (2002);“Goodman & Gilman's The Pharmaceutical Basis of Therapeutics,”McGraw-Hill, New York, 10th ed. (2001); and Remingtons's PharmaceuticalSciences, 20th Ed., 2001 can be consulted.

The DHA of the present invention is orally active and this route ofadministration can be used for the methods described herein.Accordingly, administration forms can include, but are not limited to,tablets, dragees, capsules, caplets, gelatin capsules, and pills, whichcontain the DHA and one or more suitable pharmaceutically acceptablecarriers.

Dosage forms for oral administration may include, but are not limitedto, tablets, dragees, capsules, caplets, gel caps, and pills, whichcontain the DHA and one or more suitable pharmaceutically acceptablecarriers. The DHA may be formulated readily by combining these compoundswith pharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compositions of DHA to be formulated as tablets, gelcaps, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.In some embodiments, the dosage form is a tablet, gel cap, pill orcaplet. Pharmaceutical preparations for oral use may be obtained byadding a solid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipientsinclude, but are not limited to, fillers such as sugars, including, butnot limited to, lactose, sucrose, mannitol, and sorbitol; cellulosepreparations such as, but not limited to, maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, vegetableoil (e.g., soybean oil), and polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as, but not limited to, thecross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof such as sodium alginate. Pharmaceutical preparations which maybe used orally include, but are not limited to, push-fit capsules madeof gelatin, as well as soft, sealed capsules made of gelatin (e.g., fromporcine or bovine) and a plasticizer, such as glycerol or sorbitol.Capsule shells may be composed of non-animal derived ingredients, i.e.,vegetarian ingredients, such as carrageenan, alginate, modified forms ofstarch, cellulose and/or other polysaccharides. In specific embodiments,the gelatin capsules may be porcine, bovine, vegetarian, or alginategelatin capsules. All formulations for oral administration should be indosages suitable for such administration.

In some embodiments, the dosage form is a gel cap having an amount ofDHA of about 200 mg to about 2 g, and a pharmaceutically acceptableexcipient. In some embodiments, the gel cap has an amount of DHA ofabout 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 900 mg, 1g, 1.5 g, or 2 g, and a pharmaceutically acceptable excipient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention can include othersuitable agents such as flavoring agents, preservatives, andantioxidants. In particular, it is desirable to mix the microbial oilswith an antioxidant to prevent oxidation of the DHA. Such antioxidantsare pharmaceutically acceptable and can include vitamin E, carotene, BHTor other antioxidants known to those of skill in the art.

In some embodiments, the dosage form is a nutraceutical dosage form. Theterm “nutraceutical” refers to any substance that is (1) a sole item ofa meal or diet that provides medical and/or health benefits, or (2) aproduct that is intended to supplement the diet that bears or containsone or more of the following dietary ingredients: a vitamin, a mineral,an herb or other botanical, an amino acid, a dietary substance for useby man to supplement the diet by increasing the total daily intake, or aconcentrate, metabolite, constituent, extract, or combinations of theseingredients that provides medical and/or health benefits. The medicaland/or health benefits can include reducing the risk of a neurologicaldisorder described herein.

In some embodiments, the DHA can be provided in a dietary supplement,medical food or animal feed. “Dietary supplement” refers to a compoundor composition used to supplement the diet of an animal or human. Insome embodiments, the dietary supplement can further comprise various“dietary ingredients” intended to supplement the diet. “Dietaryingredients” can further include: vitamins, minerals, herbs or otherbotanicals, amino acids, and substances such as enzymes, organ tissues,glandulars, and metabolites. Dietary ingredients can also includeextracts or concentrates. In some embodiments, the dosage form of DHA isadministered in a dietary supplement.

The present invention is also directed to use of an oral dosage formconsisting essentially of about 430 mg to about 6 g of docosahexaenoicacid (DHA) wherein the dosage form comprises less than about 1%eicosapentaenoic acid (EPA) and less than about 2% docosapentaenoic acid22:5n-6 (DPAn-6). In some embodiments, the oral dosage form is a unitdosage form, in particular, a gelatin capsule. Optionally the gelatincapsule also comprises a colorant, flavoring, and/or antioxidant.

The present invention is also directed to use of oral dosage formscomprising: (a) about 200 mg to about 4 g of DHA, wherein the DHA isabout 40% to about 99.5% (wt/wt) or more of the total fatty acid contentof the dosage foim; and (b) a pharmaceutically acceptable excipient,wherein the dosage faun is substantially free of EPA, and wherein theDHA, such as a DHA alkyl ester, is derived from an algal source.

The present invention includes gelatin capsules that are hard or softgelatin capsules. In some embodiments, the encapsulating materialcomprises a gelatin, a plasticizer, and water. In certain embodiments,the encapsulating material is vegetarian, i.e., made from non-animalderived material, including plants, seaweed (for example, carrageenan),food starch, modified corn starch, potato starch, and tapioca. In otherembodiments, the encapsulating material is derived from animals,including porcine, bovine, and fish-based materials, such as gelatins.Plasticizers of the invention include glycerin, glycerol, polyols, andmixtures thereof In some embodiments, the plasticizer is a high boilingpoint polyol, such as glycerol or sorbitol.

In some embodiments, the gelatin capsule is a soft-gelatin capsule madefrom gelatin, glycerol, and water, and filled with DHA and anantioxidant. In certain embodiments, the gelatin capsule is animal orvegetable derived. In some embodiments, the gelatin capsule comprises a0.5 g dosage form, wherein the fill weight of the weight of the dosageform is from about 450 mg to about 550 mg, and wherein the gelatincapsule comprises from about 430 mg to about 480 mg DHA. In someembodiments, the gelatin capsule comprises about 450 mg DHA per 500 mgof the dosage faun. In some embodiments, the gelatin capsule comprisesabout 450 mg DHA per 500 mg of the dosage form. In some embodiments, thegelatin capsule comprises a 1 g dosage form, wherein the fill weight ofthe dosage form is from about 950 mg to about 1050 mg, and wherein thegelatin capsule comprises from about 860 mg to about 950 mg DHA per 1000mg of the dosage form. In some embodiments, the gelatin capsulecomprises about 900 mg DHA per 1,000 g of the dosage form.

In certain embodiments, the gelatin capsule is vegetarian. In someembodiments, the capsule preparation comprises no animal products, andcomprises glycerol (and/or polyols), seaweed extract (carrageenan) andwater. In some embodiments, the water is purified. In some embodiments,color, flavor and/or sweeteners are added. During encapsulation, in someembodiments, fractionated coconut oil is used as a lubricant.

In some embodiments, the gelatin capsule comprises a capsulepreparation, an active, and optionally a colorant and/or antioxidant. Inanother embodiment i) the capsule preparation comprises gelatin (bovineacid hide), glycerin, and purified water, ii) the active comprisesDHA-EE, iii) the optional colorant is selected from titanium dioxide,FD&C Yellow #5, FD&C Red 40, and mixtures thereof; and iv) theantioxidant is ascorbyl palmitate. In some embodiments, the rawmaterials are USP raw materials.

In some embodiments, the gelatin capsules are soft gelatin capsules ofabout 1 g, having the specifications within the limits set forth inTable 8:

TABLE 8 Specifications for 1 g DHA Ethyl Ester Gelatin Capsules TESTSPECIFICATION DHA EE CONTENT, PER CAPSULE 855-945 MG AVERAGE FILL WEIGHT950-1050 MG DISINTEGRATION COMPLIES USP ACID VALUE MAX 2 MG KOH/GPEROXIDE VALUE (PV) MAX 10 MEQ/KG ANISIDINE VALUE (AV) MAX 20 MICROBIALLIMITS TESTS COMPLIES WITH <61> USP

Set forth in Table 9 is a list of components that are, in someembodiments, used in the manufacture of a DHA-EE soft gelatin capsule,and at least one corresponding function for each component.

TABLE 9 List of Components in 1 g DHA Ethyl Ester Soft Gelatin CapsulesCOMPONENT FUNCTION 900 MG DHA EE ACTIVE GELATIN, BOVINE ACID HIDECAPSULE PREPARATION GLYCERIN CAPSULE PREPARATION PURIFIED WATER CAPSULEPREPARATION TITANIUM DIOXIDE COLORANT FD&C YELLOW #5 COLORANT FD&C RED#40 COLORANT

10157] The present invention is also directed to kits or packagescomprising one or more dosage forms to be administered according to themethods described herein. A kit or package can contain one dosage form,or more than one dosage foul), (i.e., multiple dosage forms). Ifmultiple dosage forms are present in the kit or package, the multipledosage forms can be optionally arranged for sequential administration.The kits can contain dosage forms of a sufficient number to provideconvenient administration to a subject who has a chronic condition andrequires long-term administration of the DHA of the present invention.For example, in some embodiments, the kit provides dosage foams of asufficient number for 1, 2, 3 or 4 months of daily administration of theDHA. In some embodiments of the present invention, the kit comprisesdosage forms for shorter periods of administration, e.g., the kit cancontain about 7, 14, 21, 28 or more dosage fauns for oraladministration, each dosage form comprising about 450 mg to about 12.05g DHA and intended for ingestion on successive days.

The kits can optionally contain instructions associated with the dosageforms of the kits. Such instructions can be in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofthe manufacture, use or sale for human administration to treat acondition or disorder. The instructions can be in any form which conveysinformation on the use of the dosage forms in the kit according to themethods described herein. By way of example and not limitation, theinstructions can be in the form of printed matter, or in the form of apre-recorded media device.

In the course of examination of a subject, a medical professional candetermine that administration of DHA pursuant to one of the methodsdescribed herein is appropriate for the subject, or the physician candetermine that the subject's condition can be improved by theadministration of DHA pursuant to one of the methods described herein.Prior to prescribing any DHA regimen, the physician can counsel thesubject, for example, on the various risks and benefits associated withthe regimen. The subject can be provided full disclosure of all theknown and suspected risks associated with the regimen. Such counselingcan be provided verbally, as well as in written faun. In someembodiments, the physician can provide the subject with literaturematerials on the regimen, such as product information, educationalmaterials, and the like.

The present invention is also directed to methods of educating consumersabout the methods of treating neurological disorders, the methodcomprising distributing the DHA dosage forms with consumer informationat a point of sale. In some embodiments, the distribution will occur ata point of sale having a pharmacist or healthcare provider.

The term “consumer information” can include, but is not limited to, anEnglish language text, non-English language text, visual image, chart,telephone recording, website, and access to a live customer servicerepresentative. In some embodiments, consumer information will providedirections for use of the DHA unit dosages according to the methodsdescribed herein, appropriate age, use, indication, contraindications,appropriate dosing, warnings, telephone number, and website address. Insome embodiments, the method further comprises providing professionalinformation to relevant persons in a position to answer consumerquestions regarding use of the disclosed regimens according to themethods described herein. The term “professional information” includes,but is not limited to, information concerning the regimen whenadministered according to the methods of the present invention that isdesigned to enable a medical professional to answer customer questions.

A “medical professional,” includes, for example, a physician, physicianassistant, nurse practitioner, pharmacist and customer servicerepresentative. All of the various aspects, embodiments and optionsdescribed herein can be combined in any and all variations.

In some embodiments, the DHA is administered in a single dosage form,i.e., a dosage form, or in two or more dosage forms. As used herein,“dosage faun” refers to the physical form for the route ofadministration. The term “dosage form” can refer to any traditionallyused or medically accepted administrative forms, such as oraladministrative forms, intravenous administrative forms, orintraperitoneal administrative forms. In some embodiments, the DHA isadministered in a single dose, i.e., a unit dose. As used herein, a“unit dose” refers to an amount of DHA administered to a subject in asingle dose, e.g., in a gel capsule. The term “unit dose” can also referto a single unit of pharmaceutically suitable solid, liquid, syrup,beverage, or food item, that is administered within a short period oftime, e.g., within about 1 minute, 2 minutes, 3 minutes, 5 minutes, 10minutes, 20 minutes, or 30 minutes.

In some embodiments, the subject to be treated can be administered atleast one unit dose per day. In some embodiments, the dosage forms canbe taken in a single application or multiple applications per day. Forexample, if four capsules are taken daily, each capsule comprising about500 mg DHA, then all four capsules could be taken once daily, or 2capsules could be taken twice daily, or 1 capsule could be taken every 6hours. Various amounts of DHA can be in a unit dose. In someembodiments, the unit dose comprises about 430 mg, about 450 mg, about500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1g, or about 1.5 g, DHA.

In some embodiments, the dosage form has a total weight of about 0.2 gto about 2 g. By way of example and not limitation, a capsule cancontain a total weight an algal oil of about 0.2 g, where the algal oilcontain comprises DHA at a certain wt % of the total fatty acid contentof the algal oil. In some embodiments, the dosage form has a totalweight of about 0.2 g, about 0.25, about 0.3 g, about 0.35 g, about 0.4g, about 0.45 g, about 0.5 g, about 0.55 g, about 0.6 g, about 0.65 g,about 0.7 g, about 0.75 g, about 0.8 g, about 0.85 g, about 0.9 g, about0.95 g, about 1 g or about 1.05 g.

EXAMPLES

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

Effect of Pretreatment with DHA on Traumatic Brain Injury

Experimental Design. Two groups of 20 (n=40) of adult maleSprague-Dawley rats were subjected to an impact acceleration injury(IAI) resulting in reproducible severe traumatic brain injury. Ratsweighing between 350 and 400 g received induction anesthesia followed byendotracheal intubation and maintained on inhaled anesthetic using amodified medical anesthesia machine. The animals were then shaved andprepared in sterile fashion surgery, followed by subcutaneous injectionof local anesthetic into the planned incision site. A 3 cm midlineincision in the scalp was made, periosteal membranes were separated,exposing bregma and lambda. A metal disk 10 mm in diameter and 3 mmthick was attached to the skull with cyanoacrylate and centered betweenbregma and lambda. The animal were placed prone on a foam bed with themetal disk directly under a Plexiglas tube. A 450 g brass weight wasdropped through the tube from a height of 2 meters striking the disk.The animal was then ventilated on 100% O₂ while the skull was inspectedand the incision repaired. When the animal recovered spontaneousrespirations, the endotracheal tube was removed and the animal returnedto the cage for postoperative observation (Foda and Marmarou, JNeurosurg 80:301-313 (1994)). All procedures involving live animals havebeen previously reviewed and approved by the Institutional Animal Careand Use Committee of West Virginia University, and were performedaccording to the principles of the Guide for the Care and Use ofLaboratory animals, published by the Institute of Laboratory Resources,National Research Council (NIH publication 85-23-2985).

Animals and dietary treatments. The two treatment groups were housed inthe small animal vivarium under veterinary staff supervision for 40days. At weaning age (21 days) pre-injury, the groups received daily acontrol diet or that same diet supplemented with algae derived DHA. Eachgroup received rat chow ad lib.

After 28 days on the diets, treatment groups underwent IAI procedure.Following IAI, all dietary groups were fed the control diet (standardchow diet, without added DHA).

Immunohistochemical analyses. Following 7 days post-injury survival,half of the animals in each group were euthanized with a lethal doseinjection of 00.5 ml Ketamine and 0.5 ml Xylazine. The animals wereimmediately perfused transcardially with 200 ml cold 0.9% saline to washout all blood. This was followed by 4% paraformaldehyde in Millonigsbuffer for 40 minutes. The entire brain, brainstem, and rostral spinalcord were removed and immediately placed in 4% parafonnaldehyde for 24hours. Following fixation, the brain was blocked by cutting thebrainstem above the pons, cutting the cerebellar peduncies, and thenmaking sagittal cuts lateral to the pyramids. The resulting tissuecontaining the corticospinal tracts and the medial lemnisci, areas shownpreviously to yield traumatically injured axons, was then sagitally cuton a vibratome into 40 micron sections. The tissue then underwenttemperature controlled microwave antigen retrieval using previouslydescribed techniques (Stoneet al., Acta Neuropathol 97:335-345 (1999)).Briefly, the tissue was preincubated in a solution containing 10% normalgoat serum (NGS) and 0.2% triton X in PBS for 40 minutes.

The tissue was incubated in polyclonal antibody raised in rabbit againstbeta amyloid precursor protein (B-APP) at a dilution of 1:200 in 1% NGSin PBS, then incubated in a secondary anti-rabbit IgG antibodyconjugated with Alexa 488 fluorophore for two hours. The tissueunderwent a final wash in 0.1M phosphate buffer and then was mountedusing an antifade agent and coverslip. The slides were sealed withacrylic and stored in the dark in a laboratory refrigerator (Mills etal., J Biomed Opt 8:347-356 (2003)). A similar histological approach wasused to assess microglia/macrophage recruitment into the injured brainregions, neuron and oligodendrocyte survival (Huang et al., Brain130:3004-3019 (2007)).

The tissue was then examined and images acquired using a laser scanningconfocal microscope system and a 40× objective lens. Ten digital imagesare obtained from the tissue of each animal, and images are thenrandomized. Individual injured axons are independently counted and datais stored in an Excel spreadsheet, Statistical analysis of the data isperformed using Statistical software.

Behavioral assessment. Fourteen days post-injury, functional evaluationof learning and memory was performed on all four groups. This consistedof pre- and post-injury testing using a Morris Watermaze task (Stone etal., Acta Neuropathol 97:335-345 (1999)). Fatty acid blood testing wasdone prior to the injury and at the end of study.

Animals (rats) were given 0, 3, 9, 40 mg/kg/day of DHASCO oil containingDHA for 28 days prior to inducing traumatic brain injury. Results of thestudy are shown in Table 10 below.

TABLE 10 Sham None 3 mg/kg 9 mg/kg 40 mg/kg Axon damage (%)  2 100  7564 15 Neuronal death (%) 13 100  73 77 22 Inflammation 10 100  74 71 31Water maze navigation 22 110 100 94 32 (sec)

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1. A method for reducing the risk of pathological effects of traumaticbrain injury, comprising: (a) administering to a subject who is at riskof traumatic brain injury a composition comprising at least about 35 wt% docosahexaenoate (DHA), wherein the composition is administered in aprophylactically effective amount for a sufficient time period prior toengagement in an activity associated with a risk of traumatic braininjury to reduce the risk of pathological effects of traumatic braininjury, and wherein the composition has an eicosapentaenoate (EPA)content of less than about 2 wt % of the total fatty acid content. 2.(canceled)
 3. The method of claim 1 in which the DHA is in the form of atriglyceride.
 4. The method of claim 3 in which the DHA is in the formof an alkylester.
 5. The method of claim 4 in which the DHA alkylesteris a ethyl ester.
 6. The method of claim 5 in which the DHA alkylesteris at least about 85 wt % of the total fatty acid content of thecomposition.
 7. The method of claim 6 in which the DHA alkylester isabout 85 to about 96 wt % of the total fatty acid content of thecomposition.
 8. The method of claim 1 in which the DHA is at least about40 wt % of the total fatty acid content of the composition.
 9. Themethod of claim 8 in which the DHA is about 40 to about 50 wt % of thetotal fatty acid content of the composition.
 10. The method of claim 1in which the DHA is at least about 55 wt % of the total fatty acidcontent of the composition.
 11. The method of claim 10 in which the DHAis about 55 to 65 wt % of the total fatty acid content of thecomposition.
 12. The method of claim 1 in which the DHA to EPA ratio isat least 10:1.
 13. The method of claim 1 in which the DHA to EPA ratiois at least 100:1. 14-17. (canceled)
 18. The method of claim 1 in whichthe traumatic brain injury is from a closed head injury. 19-21.(canceled)
 22. The method of claim 1 in which the composition isadministered for at least 28 days prior to engaging in the activityassociated with a risk of traumatic brain injury.
 23. The method ofclaim 1 in which the composition is administered for at least 6 weeksprior to engaging in the activity associated with a risk of fortraumatic brain injury.
 24. The method of claim 1 in which the effectiveamount is a dose of about 10 mg/kg body weight/day to about 40 mg/kgbody weight/day of DHA.
 25. The method of claim 1 in which thecomposition is an oral dosage form.
 26. The method of claim 25 in whichthe oral dosage form is a gelatin capsule.
 27. The method of claim 26 inwhich the gelatin capsule comprises from about 200 mg to about 1 g ofDHA, and a pharmaceutically acceptable excipient.
 28. (canceled)
 29. Amethod of protecting the brain of a human subject, the methodcomprising: administering to the subject, before an activity associatedwith a potential traumatic brain injuring event, an oral dosage formcomprising at least 900 mg of DHA, wherein the dosage form comprises atleast about 35 wt % docosahexaenoate (DHA) of the total fatty acidcontent, wherein the dosage form has an eicosapentaenoate (EPA) contentof less than about 2 wt % of the total fatty acid content.
 30. Themethod of claim 29, wherein the activity associated with a potentialtraumatic brain injuring event is selected from the group consisting ofboxing, football, soccer, hockey, aimed conflict, or brain surgery. 31.The method of claim 29, wherein protecting the brain of a subject is theprevention of the pathological effects of a concussion, or the reductionof the pathological effects associated with a concussion
 32. (canceled)